Humanized or chimeric cd3 antibodies

ABSTRACT

The present invention relates to humanized or chimeric antibodies binding CD3. It furthermore relates to bispecific antibodies, compositions, pharmaceutical compositions, use of said antibodies in the treatment of a disease, and method of treatment.

FIELD OF INVENTION

The present invention relates to a humanized or chimeric antibodybinding to human CD3, compositions comprising said humanized or chimericantibody, and use of said humanized or chimeric antibodies in treatmentof a disease.

BACKGROUND

The Cluster of Differentiation 3 (CD3) has been known for many years andtherefore has been subject of interest in many aspects. Specificallyantibodies raised against CD3 or the T-cell Receptor Complex, which CD3is part of, are known. An in vitro characterization of five humanizedOKT3 effector function variant antibodies has been described [1].

Treatment with the anti-CD3 monoclonal antibody hOKT3 gamma1(Ala-Ala)results in improved C-peptide responses and clinical parameters for atleast 2 years after onset of type 1 diabetes in absence of continuedimmunosuppressive medications [2].

A promising approach to improve targeted antibody therapy is bydelivering cytotoxic cells specifically to the antigen-expressing cancercells. This concept of using T-cells for efficient killing of tumorcells has been described [3]. However, initial clinical studies wererather disappointing mainly due to low efficacy, severe adverse effects(cytokine storm) and immunogenicity of the bispecific antibodies [4].Advances in the design and application of bispecific antibodies havepartially overcome the initial barrier of cytokine storm and improvedclinical effectiveness without dose-limiting toxicities [5].

For example, certain bispecific antibodies targeting with one arm theantigen on the tumor cell and with the other arm for instance CD3 on Tcells, provide Fc receptor binding by the Fc region. Upon binding, acomplex of T cells, tumor cells and effector cells that bind theantibody Fc region is formed, leading to killing of the tumor cells [4].Catumaxomab consists of a mouseIgG2a/ratIgG2b heterodimer and has beenfound successful for the treatment of cancer-associated ascites afterintraperitoneal application [6]. However, the mouse/rat hybrid isimmunogenic [7] and cannot be applied for long-term intravenoustreatment in humans. Frequent treatment-related adverse eventsattributed to catumaxomab included cytokine-release-related symptoms(i.e. pyrexia, nausea, vomiting, chills, tachycardia and hypotension)[8]-[9], which relate to the effector functions of the Fc region ofcatumaxomab. Another antibody is ertumaxomab (HER2×CD3), which inducescytotoxicity in cell lines with low HER2 expression. Ertumaxomab hasbeen in Phase II clinical development for metastatic breast cancer[10]-[11].

CD3 antibodies cross-reactive to cynomolgus and/or rhesus monkey CD3have been described [12]-[13], however, further improvements for suchcross-reactive antibodies are needed.

SUMMARY OF INVENTION

It is an object of the present invention to provide humanized orchimeric CD3 antibodies. Thus, in one aspect, the present inventionrelates to a humanized or chimeric antibody binding to human CD3,wherein said antibody comprises a binding region comprising heavy chainvariable (VH) region CDR1, CDR2, and CDR3 having the sequences as setforth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable(VL) region CDR1, CDR2, and CDR3 having the sequences as set forth inSEQ ID NO: 4, the sequence GTN, and the sequence as set forth in SEQ IDNO: 5 or SEQ ID NO:60, respectively.

in one aspect, the present invention relates to a humanized or chimericantibody binding to human CD3, wherein said antibody comprises a bindingregion comprising heavy chain variable (VH) region CDR1, CDR2, and CDR3having the sequences as set forth in SEQ ID NOs: 1, 2, and 3,respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3having the sequences as set forth in SEQ ID NO: 4, the sequence GTN, andthe sequence as set forth in SEQ ID NO: 5, respectively.

In another aspect, the present invention relates to a bispecificantibody comprising a first binding region of an antibody according tothe invention, and a second binding region which binds a differenttarget than said first antigen binding region.

In another aspect, the present invention relates to a nucleic acidconstruct encoding one or more amino acid sequences according to theinvention.

In another aspect, the present invention relates to an expression vectorcomprising (i) a nucleic acid sequence encoding a heavy chain sequenceof a humanized or chimeric antibody according to the invention, (ii) anucleic acid sequence encoding a light chain sequence of a humanized orchimeric antibody according to the invention, or (iii) both (i) and(ii).

In another aspect, the present invention relates to a host cellcomprising an expression vector according to the invention.

In another aspect, the present invention relates to a compositioncomprising the antibody or bispecific antibody according to theinvention.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising the antibody or bispecific antibody according tothe invention and a pharmaceutical acceptable carrier

In another aspect, the present invention relates to the antibody orbispecific antibody, the composition, or the pharmaceutical compositionaccording to the invention for use as a medicament.

In another aspect, the present invention relates to the antibody orbispecific antibody, the composition, or the pharmaceutical compositionaccording to the invention for use in the treatment of a disease.

In another aspect, the present invention relates to a method oftreatment of a disease comprising administering the antibody orbispecific antibody, the composition, or the pharmaceutical compositionaccording to the invention, to a subject in need thereof.

In one aspect, the present invention relates to a method of diagnosing adisease characterized by involvement or accumulation of CD3-expressioncells, comprising administering the humanized or chimeric antibody, thecomposition or the pharmaceutical composition according to the inventionto a subject, optionally wherein said humanized or chimeric antibody islabeled with a detectable agent.

In another aspect, the present invention relates to a method forproducing an antibody or a bispecific antibody according to theinvention, comprising the steps of a) culturing a host cell according tothe invention, and b) purifying the antibody from the culture media.

In another aspect, the present invention relates to a diagnosticcomposition comprising an antibody or bispecific antibody according tothe invention.

In another aspect, the present invention relates to a method fordetecting the presence of CD3 antigen, or a cell expressing CD3, in asample comprising the steps of a) contacting the sample with an antibodyor bispecific antibody according to the invention, under conditions thatallow for formation of a complex between the antibody or bispecificantibody and CD3, and b) analyzing whether a complex has been formed.

In another aspect, the present invention relates to a kit for detectingthe presence of CD3 antigen, or a cell expressing CD3, in a samplecomprising i) an antibody or bispecific antibody according to theinvention, and ii) instructions for use of the kit.

In another aspect, the present invention relates to an anti-idiotypicantibody which binds to an antibody according to the invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Shows binding curves of (FIG. 1A) monospecific antibody variantsof IgG1-huCD3 and (FIG. 1B) bispecific antibody variants bsIgG1huCD3×HER2 to the human T-cell line Jurkat. Data shown are meanfluorescence intensities (MFI) of one representative experiment, asdescribed in Example 2. The tables show the antibody concentrations(μg/mL) that result in half-maximal binding (EC50).

FIG. 2: Shows binding curves of (FIG. 2A) monospecific antibody variantsof IgG1-huCD3 and (FIG. 2B) bispecific antibody variants bsIgG1huCD3×HER2 to the cynomolgus T-cell line HSC-F. Data shown are meanfluorescence intensities (MFI) of one representative experiment, asdescribed in Example 2.

FIG. 3: T-cell activation by IgG1-huCD3 antibody variants. Expression ofCD69 on T-cells from human (FIG. 3A) and cynomolgus (FIG. 3B) origin inPBMC culture was measured by FACS analysis, as described in Example 3.These experiments were performed twice and representative results fromone experiment are shown.

FIG. 4: T-cell proliferation induced by IgG1-huCD3 antibody variants.Human (FIG. 4A) or cynomolgus (FIG. 4B) PBMCs were incubated withIgG1-huCD3 antibody variants for 3 days, after which proliferation wasmeasured by a cell proliferation ELISA, as described in Example 4.Representative results from two independent experiments are shown.

FIG. 5: Induction of human (FIG. 5A) and cynomolgus (FIG. 5B)T-cell-mediated cytotoxicity by huCD3 antibody variants withnon-activating LFLEDA mutations were determined as explained in Example5. Representative results from two independent experiments performed induplo are shown.

FIG. 6: Shows binding curves of non-activating, monospecific antibodyvariants of IgG1-huCD3 (FIG. 6A) and non-activating, bispecific antibodyvariants bsIgG1-huCD3×HER2 (FIG. 6B) to the human T-cell line Jurkat.Data shown are mean fluorescence intensities (MFI) of one representativeexperiment, as described in Example 2. The tables show the antibodyconcentrations (μg/mL) that result in half-maximal binding (EC50).

FIG. 7: Shows binding curves of non-activating, monospecific antibodyvariants of IgG1-huCD3 (FIG. 7A) and non-activating, bispecific antibodyvariants bsIgG1-huCD3×HER2 (FIG. 7B) to the cynomolgus T-cell lineHSC-F. Data shown are mean fluorescence intensities (MFI) of onerepresentative experiment, as described in Example 2. The tables showthe antibody concentrations (μg/mL) that result in half-maximal binding(EC50).

FIG. 8: T-cell activation by non-activating monospecific IgG1-huCD3(FIGS. 8A and B) or non-activating bispecific bsIgG1-huCD3×HER2 antibodyvariants (FIGS. 8C and D). Expression of CD69 on T-cells from human(FIGS. 8A and C) and cynomolgus (FIGS. 8B and D) origin in PBMC culturewas measured by FACS analysis, as described in Example 3. Theseexperiments were performed twice and representative results from oneexperiment are shown.

FIG. 9: T-cell proliferation induced by non-activating monospecificIgG1-huCD3 (FIGS. 9A and B) or non-activating bispecificbsIgG1-huCD3×HER2 antibody variants (FIGS. 9C and D). T-cellproliferation was measured in human (FIGS. 9A and C) or cynomolgus(FIGS. 9B and D) PBMCs that were incubated with various antibodyvariants for 3 days, after which proliferation was measured by a cellproliferation ELISA, as described in Example 4. Representative resultsfrom two independent experiments are shown.

FIG. 10: Induction of human (FIG. 10A) and cynomolgus (FIG. 10B)T-cell-mediated cytotoxicity by huCD3 antibody variants withnon-activating LFLEDA mutations were determined as explained in Example5. Representative results from two independent experiments performed induplo are shown.

FIG. 11: Rhesus T-cell activation by IgG1-huCD3 antibody variants.Expression of CD69 on T-cells from rhesus origin in PBMC culture wasmeasured by FACS analysis, as described in Example 6.

FIG. 12: T-cell activation by non-activating variants of huCLB-T3/4antibody. IgG1-huCLB-T3/4 variants were titrated on PBMCs. Expression ofCD69 on T-cells in PBMC culture was measured by FACS analysis, asdescribed in Example 7. Representative examples of 3 experiments areshown.

FIG. 13: T-cell proliferation by non-activating variants of huCLB-T3/4antibody. PBMCs were incubated with antibodies for three days, afterwhich proliferation was measured by a cell proliferation ELISA, asdescribed in Example 8. Representative results from two independentexperiments are shown.

FIG. 14: In vitro T-cell-mediated cytotoxicity induced by non-activatingantibody variants of a CD3 antibody. Induction of T-cell-mediatedcytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ,LFLEDANQPS [FIG. 14A-G]) was determined as explained in Example 9. Theaverages from two experiments performed in duplo are shown.

FIG. 15: In vitro T-cell mediated cytotoxicity induced by non-activatinghuCLB-T3/4 variants. Induction of T-cell mediated cytotoxicity byantibody variants (LFLEDA LAL [FIG. 15A-C] was determined as describedin Example 9. The averages from one experiment performed in duplet areshown.

FIG. 16: Evaluation of binding of C1q to non-activating huCLB-T3/4antibody variants. Binding of C1q to monospecific IgG1 huCLB-T3/4 (FIG.16A-C) and bsIgG1-huCLB-T3/4×HER2 (FIG. B-D) and non-activating antibodyvariants thereof was evaluated by ELISA as described in Example 10. Theresults in the graphs are representative for n=2 experiments.

FIG. 17: Pharmacokinetic (PK) analysis of non-activating huCLB-T3/4antibody variants were compared to that of wild-type IgG1-huCLB-T3/4antibody as described in Example 11. Plasma concentration of human IgG1was plotted against time (FIG. 17A). Plasma clearance rate calculated asdescribed in Example 11 (FIG. 17B). The horizontal dotted linerepresents the average clearance rate of human IgG1 antibodies in SCIDmice (10 mL/day/kg).

FIG. 18: The frequency of positive T-cell responses among healthyHLA-typed donors. SI indexes of ≧1.9 in both proliferation and IL-2secretion assays were considered positive responses. Humanized A33 wasused as clinical benchmark control antibody that shows high level ofimmunogenicity in the clinic and routinely induces 20-30% T-cellresponses in the EpiScreen Assay. KLH responses were included to checkPBMC quality (after thawing).

FIG. 19: Sequence alignment of heavy chain (VH) and light chain (VL)variable regions of humanized CD3 antibodies according to the presentinvention.

FIG. 20: Expression levels of IgG1-huCD3-H1L1 variants in Expi293Fsupernatants as measured by Octet RED using anti human IgG immobilizedsensors.

FIG. 21: Binding of IgG1-huCD3-H1L1-LFLEDA mutants to Jurkat cells.

FIG. 22: Binding of the CD3 bispecific antibody variants to human Tcells. The circles depict the binding of the huCD3-H1L1-LFLEDA variants,the triangles depict the binding of the huCD3-H1L1-LFLEDA-LK55N variantsand the squares depict the binding of the CD3-hmAb286-LFLEDA variants.

FIG. 23: Cytotoxicity of huCD3×HER2 antibody affinity variants on A431cells (low HER2 expressing cells) (FIG. 23A) and AU565 cells (high HER2expressing cells) (FIG. 23B) treated with CD3 and HER2 affinityvariants.

FIG. 24: Reduction of tumor size in NOD-SCID mice byhuCD3-H1L1×HER2-LFLEDA 7, 14, 21 and 28 days after tumor inoculation(FIG. 24A), and at day 29 (FIG. 24B).

FIG. 25: Reduction of tumor size in NOD-SCID mice byhuCD3-H1L1×CD20-LFLEDA 7, 14, and 21 days after tumor inoculation (FIG.25A), and at day 21 (FIG. 25B).

DETAILED DESCRIPTION

In one aspect, the present invention relates to a humanized or chimericantibody binding to human CD3, wherein said antibody comprises a bindingregion comprising heavy chain variable (VH) region CDR1, CDR2, and CDR3having the sequences as set forth in SEQ ID NOs: 1, 2, and 3,respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3having the sequences as set forth in SEQ ID NO: 4, the sequence GTN, andthe sequence as set forth in SEQ ID NO: 5 or SEQ ID NO:60, respectively.

In one embodiment, the present invention relates to a humanized orchimeric antibody binding to human CD3, wherein said antibody comprisesa binding region comprising heavy chain variable (VH) region CDR1, CDR2,and CDR3 having the sequences as set forth in SEQ ID NOs: 1, 2, and 3,respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3having the sequences as set forth in SEQ ID NO: 4, the sequence GTN, andthe sequence as set forth in SEQ ID NO: 5, respectively.

The term “antibody” as used herein is intended to refer to animmunoglobulin molecule, a fragment of an immunoglobulin molecule, or aderivative of either thereof, which has the ability to specifically bindto an antigen under typical physiological conditions with a half-life ofsignificant periods of time, such as at least about 30 minutes, at leastabout 45 minutes, at least about one hour, at least about two hours, atleast about four hours, at least about 8 hours, at least about 12 hours,about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 ormore days, etc., or any other relevant functionally-defined period (suchas a time sufficient to induce, promote, enhance, and/or modulate aphysiological response associated with antibody binding to the antigenand/or time sufficient for the antibody to recruit an effectoractivity). The binding region (or binding domain which may also be usedherein, both terms having the same meaning) which interacts with anantigen, comprises variable regions of both the heavy and light chainsof the immunoglobulin molecule. The constant regions of the antibodies(Abs) may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (such as effectorcells and T-cells) and components of the complement system such as C1q,the first component in the classical pathway of complement activation.As indicated above, the term antibody as used herein, unless otherwisestated or clearly contradicted by context, includes fragments of anantibody that retain the ability to specifically interact, such as bind,to the antigen. It has been shown that the antigen-binding function ofan antibody may be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term “antibody”include (i) a Fab′ or Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(H), C_(L) and C_(H)1 domains, or a monovalent antibody asdescribed in WO2007059782 (Genmab A/S); (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting essentially of theV_(H) and C_(H)1 domains; and (iv) a Fv fragment consisting essentiallyof the V_(L) and V_(H) domains of a single arm of an antibody.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they may be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain antibodies or singlechain Fv (scFv), see for instance Bird et al., Science 242, 423-426(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such singlechain antibodies are encompassed within the term antibody unlessotherwise noted or clearly indicated by context. Although such fragmentsare generally included within the meaning of antibody, they collectivelyand each independently are unique features of the present invention,exhibiting different biological properties and utility. These and otheruseful antibody fragments in the context of the present invention arediscussed further herein. It also should be understood that the termantibody, unless specified otherwise, also includes polyclonalantibodies, monoclonal antibodies (mAbs), chimeric antibodies andhumanized antibodies, and antibody fragments retaining the ability tospecifically bind to the antigen (antigen-binding fragments) provided byany known technique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype.

The term “immunoglobulin heavy chain”, “heavy chain of animmunoglobulin” or “heavy chain” as used herein is intended to refer toone of the chains of an immunoglobulin. A heavy chain is typicallycomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region (abbreviated herein as CH) whichdefines the isotype of the immunoglobulin. The heavy chain constantregion typically is comprised of three domains, CH1, CH2, and CH3. Theheavy chain constant region may further comprise a hinge region. Theterm “immunoglobulin” as used herein is intended to refer to a class ofstructurally related glycoproteins consisting of two pairs ofpolypeptide chains, one pair of light (L) low molecular weight chainsand one pair of heavy (H) chains, all four potentially inter-connectedby disulfide bonds. The structure of immunoglobulins has been wellcharacterized (see for instance [14]). Within the structure of theimmunoglobulin, the two heavy chains are inter-connected via disulfidebonds in the so-called “hinge region”. Equally to the heavy chains eachlight chain is typically comprised of several regions; a light chainvariable region (abbreviated herein as VL) and a light chain constantregion (abbreviated herein as CL). The light chain constant regiontypically is comprised of one domain, CL. Furthermore, the VH and VLregions may be further subdivided into regions of hypervariability (orhypervariable regions which may be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Each VH and VL is typically composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (see [15]). CDR sequences may be determined by use of themethod provided by IMGT [16]-[17].

The term “isotype” as used herein, refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or anyallotype thereof, such as IgG1m(za) and IgG1m(f) [SEQ ID NO:15]) that isencoded by heavy chain constant region genes. Thus, in one embodiment,the antibody comprises a heavy chain of an immunoglobulin of the IgG1class or any allotype thereof. Further, each heavy chain isotype can becombined with either a kappa (κ) or lambda (λ) light chain.

The term “chimeric antibody” as used herein, refers to an antibodywherein the variable region is derived from a non-human species (e.g.derived from rodents) and the constant region is derived from adifferent species, such as human. Chimeric antibodies may be generatedby antibody engineering. “Antibody engineering” is a term used genericfor different kinds of modifications of antibodies, and which is awell-known process for the skilled person. In particular, a chimericantibody may be generated by using standard DNA techniques as describedin [18]. Thus, the chimeric may be a genetically or an enzymaticallyengineered recombinant antibody. It is within the knowledge of theskilled person to generate a chimeric antibody, and thus, generation ofthe chimeric antibody according to the present invention may beperformed by other methods than described herein. Chimeric monoclonalantibodies for therapeutic applications are developed to reduce antibodyimmunogenicity. They may for typically contain non-human (e.g. murine)variable regions, which are specific for the antigen of interest, andhuman constant antibody heavy and light chain domains. The terms“variable region” or “variable domains” as used in the context ofchimeric antibodies, refers to a region which comprises the CDRs andframework regions of both the heavy and light chains of theimmunoglobulin.

The term “humanized antibody” as used herein, refers to a geneticallyengineered non-human antibody, which contains human antibody constantdomains and non-human variable domains modified to contain a high levelof sequence homology to human variable domains. This can be achieved bygrafting of the six non-human antibody complementarity-determiningregions (CDRs), which together form the antigen binding site, onto ahomologous human acceptor framework region (FR) (see [19]-[20]). Inorder to fully reconstitute the binding affinity and specificity of theparental antibody, the substitution of framework residues from theparental antibody (i.e. the non-human antibody) into the human frameworkregions (back-mutations) may be required. Structural homology modelingmay help to identify the amino acid residues in the framework regionsthat are important for the binding properties of the antibody. Thus, ahumanized antibody may comprise non-human CDR sequences, primarily humanframework regions optionally comprising one or more amino acidback-mutations to the non-human amino acid sequence, and fully humanconstant regions. Optionally, additional amino acid modifications, whichare not necessarily back-mutations, may be applied to obtain a humanizedantibody with preferred characteristics, such as affinity andbiochemical properties.

The humanized or chimeric antibody according to any aspect or embodimentof the present invention may be termed “humanized or chimeric CD3antibody”, “humanized or chimeric antibody of the invention”, “CD3antibody”, or “CD3 antibody of the invention”, which all have the samemeaning and purpose unless otherwise contradicted by context.

The amino acid sequence of an antibody of non-human origin is distinctfrom antibodies of human origin, and therefore a non-human antibody ispotentially immunogenic when administered to human patients. However,despite the non-human origin of the antibody, its CDR segments areresponsible for the ability of the antibody to bind to its targetantigen and humanization aims to maintain the specificity and bindingaffinity of the antibody. Thus, humanization of non-human therapeuticantibodies is performed to minimize its immunogenicity in man while suchhumanized antibodies at the same time maintain the specificity andbinding affinity of the antibody of non-human origin.

The term “binding region” as used herein, refers to a region of anantibody which is capable of binding to any molecule, such as apolypeptide, e.g. present on a cell, bacterium, or virion.

The term “binding” as used herein, refers to the binding of an antibodyto a predetermined antigen or target to which binding typically is withan affinity corresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less,about 10⁻¹⁹ M or less, or about 10⁻¹¹M or even less when determined byfor instance surface plasmon resonance (SPR) technology in a BIAcore3000 instrument using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100 fold lower, for instance at least 1,000 fold lower, such as atleast 10,000 fold lower, for instance at least 100,000 fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen. Thedegree with which the affinity is lower is dependent on the K_(D) of theantibody, so that when the K_(D) of the antibody is very low (that is,the antibody is highly specific), then the degree with which theaffinity for the antigen is lower than the affinity for a non-specificantigen may be at least 10,000 fold. The term “K_(D)” (M), as usedherein, refers to the dissociation equilibrium constant of a particularantibody-antigen interaction.

The term “human CD3” as used herein, refers to the human Cluster ofDifferentiation 3 protein which is part of the T-cell co-receptorprotein complex and is composed of four distinct chains. CD3 is alsofound in other species, and thus, the term “CD3” may be used herein andis not limited to human CD3 unless contradicted by context. In mammals,the complex contains a CD3γ (gamma) chain (human CD3γ chain SwissprotP09693, or cynomolgus monkey CD3γ Swissprot Q95LI7), a CD3δ (delta)chain (human CD3δ Swissprot P04234, or cynomolgus monkey CD3δ SwissprotQ95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766; orcynomolgus CD3ε Swissprot Q95LI5), rhesus CD3ε (Swissprot G7NCB9), and aCD3ζ-chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkeyCD3ζ Swissprot Q09TK0). These chains associate with a molecule known asthe T-cell receptor (TCR) and generate an activation signal in Tlymphocytes. The TCR and CD3 molecules together comprise the TCRcomplex.

It is within the knowledge of the skilled person that amino acidsequences referred to as Swissprot numbers include a signal peptidewhich is removed after translation of the protein. Thus, proteins, suchas CD3, present on cell surfaces do not include the signal peptide. Inparticular, the amino acid sequences listed in Table 1 do not containsuch signal peptide. Such proteins as listed in Table 1 may be termed“mature proteins”. Thus, SEQ ID NO:14 shows the amino acid sequence ofmature human CD3δ (delta), SEQ ID NO:13 shows the amino acid sequence ofmature human CD3ε (epsilon), SEQ ID NO:21 shows the amino acid sequenceof mature cynomolgus CD3ε, and SEQ ID NO:22 shows the amino acidsequence of mature rhesus CD3ε. Thus, the term “mature” as used herein,refers to a protein which does not comprise any signal or leadersequence.

It is well-known that signal peptide sequence homology, length, and thecleavage site position, varies significantly between different proteins.Signal peptides may be determined by different methods, e.g. SEQ IDNO:13 of the present invention has been determined according to theSignalP application (available onhttp://www.cbs.dtu.dk/services/SignalP/).

In a particular embodiment, the humanized or chimeric antibody of thepresent invention binds the epsilon chain of CD3, such as the epsilonchain of human CD3 (SEQ ID NO:13). In yet another particular embodiment,the humanized or chimeric antibody binds an epitope within amino acids1-27 of the N-terminal part of human CD3ε (epsilon) (SEQ ID NO:13). Insuch a particular embodiment, the antibody may even further cross-reactwith other non-human primate species, such as cynomolgus monkeys(cynomolgus CD3 epsilon SEQ ID NO:21) and/or rhesus monkeys (rhesus CD3epsilon SEQ ID NO:22).

The term “cross-react” as used herein, refers to the ability of anantibody, such as a humanized or chimeric antibody according to theinvention, to bind its target on different species. In particular, thehumanized CD3 antibody exemplified in the examples described herein, hasthe ability to both bind human (Example 2), cynomolgus (Example 2) andrhesus monkey CD3.

An antibody according to the present invention comprising the CDRsequences as defined herein, further comprising framework regions maydiffer in sequence outside the CDR sequences but still retains the fullbinding ability as compared the original antibody. Thus, the presentinvention also relates to antibodies comprising an amino acid sequenceof the variable region having a certain sequence identity to anysequence herein described.

The term “sequence identity” as used in the context of the presentinvention, refers to the percent identity between two sequences as afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The percent identity between two nucleotide or amino acid sequences maye.g. be determined using the algorithm of E. Meyers and W. Miller [21].In addition, the percent identity between two amino acid sequences maybe determined using the Needleman and Wunsch algorithm [22]. Multiplealignments are preferably performed using the Clustal W algorithm [23](as used e.g., in Vector NTI Advance® software version 11.5; InvitrogenInc.).

Thus, in one embodiment, the VH region has at least 90%, at least 95%,at least 97%, or at least 99% amino acid sequence identity to at leastone amino acid sequence as set forth in the VH sequences selected fromthe group consisting of:

a) a VH sequence as set forth in SEQ ID NO:6;

b) a VH sequence as set forth in SEQ ID NO:8;

c) a VH sequence as set forth in SEQ ID NO:7; and

d) a VH sequence as set forth in SEQ ID NO:9.

In one particular embodiment, the VH region has at least 96% amino acidsequence identity to at least one amino acid sequence as set forth inthe VH sequences selected from the group consisting of:

a) a VH sequence as set forth in SEQ ID NO:6;

b) a VH sequence as set forth in SEQ ID NO:8;

c) a VH sequence as set forth in SEQ ID NO:7; and

d) a VH sequence as set forth in SEQ ID NO:9.

In one embodiment, the VL region has at least 90%, at least 95%, atleast 97%, or at least 99% amino acid sequence identity to at least oneamino acid sequence as set forth in the VL sequences selected from thegroup consisting of:

a) a VL sequence as set forth in SEQ ID NO:10;

b) a VL sequence as set forth in SEQ ID NO:11; and

c) a VL sequence as set forth in SEQ ID NO:12.

In one particular embodiment, the VL region has at least 95% amino acidsequence identity to at least one amino acid sequence as set forth inthe VL sequences selected from the group consisting of:

a) a VL sequence as set forth in SEQ ID NO:10;

b) a VL sequence as set forth in SEQ ID NO:11; and

c) a VL sequence as set forth in SEQ ID NO:12.

In one embodiment, the VH region is selected from the group consistingof:

a) a VH sequence as set forth in SEQ ID NO:6;

b) a VH sequence as set forth in SEQ ID NO:8;

c) a VH sequence as set forth in SEQ ID NO:7; and

d) a VH sequence as set forth in SEQ ID NO:9.

In one embodiment, the VL region is selected from the group consistingof:

a) a VL sequence as set forth in SEQ ID NO:10;

b) a VL sequence as set forth in SEQ ID NO:11; and

c) a VL sequence as set forth in SEQ ID NO:12.

In one embodiment, only one of either the VH or VL sequence is 100%identical to one of the sequences disclosed herein whereas the other mayhave a sequence identity of at least 90%, at least 95%, at least 97%, orat least 99% amino acid sequence identity with one of the sequencesherein disclosed.

In one particular embodiment, the VH sequence has at least 97% aminoacid sequence identity to at least one amino acid sequence as set forthin the VH sequences selected from the group consisting of:

a) a VH sequence as set forth in SEQ ID NO:6;

b) a VH sequence as set forth in SEQ ID NO:7;

c) a VH sequence as set forth in SEQ ID NO:8; and

d) a VH sequence as set forth in SEQ ID NO:9;

and the VL sequence has at least 95% amino acid sequence identity to atleast one amino acid sequence as set forth in the VL sequences selectedfrom the group consisting of:

i. a VL sequence as set forth in SEQ ID NO: 10;

ii. a VL sequence as set forth in SEQ ID NO:11; and

iii. a VL sequence as set forth in SEQ ID NO:12.

In one embodiment, the VH and VL sequences are selected from the groupconsisting of;

a) a VH and a VL sequence having at least 90% identity to the sequencesset forth in SEQ ID NOs:6 and 10, respectively; 7 and 10, respectively;8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively;7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively;6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively;and 9 and 12, respectively;

b) a VH and a VL sequence having at least 95% identity to the sequencesset forth in SEQ ID NOs:6 and 10, respectively; 7 and 10, respectively;8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively;7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively;6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively;and 9 and 12, respectively;

c) a VH and a VL sequence having at least 97% identity to the sequencesset forth in SEQ ID NOs:6 and 10, respectively; 7 and 10, respectively;8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively;7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively;6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively;and 9 and 12, respectively;

d) a VH and a VL sequence having at least 99% identity to the sequencesset forth in SEQ ID NOs:6 and 10, respectively; 7 and 10, respectively;8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively;7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively;6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively;and 9 and 12, respectively;

e) a VH and a VL sequence having at least 100% identity to the sequencesset forth in SEQ ID NOs:6 and 10, respectively; 7 and 10, respectively;8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively;7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively;6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively;and 9 and 12, respectively;

f) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

g) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

h) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

i) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

j) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

k) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

l) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

m) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

n) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

o) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

p) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

q) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

r) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

s) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

t) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

u) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

v) a VH sequence having at least 100% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

x) a VH sequence having at least 100% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

y) a VH sequence having at least 100% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

z) a VH sequence having at least 100% identity to the sequence set forthin SEQ ID NO:6 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

aa) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ab a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ac) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ad) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ae) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

af) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ag) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ah) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ai) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

aj) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ak) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

al) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

am) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

an) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ao) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ap) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:7 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

aq) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:7 and a VL sequence having at least 90% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

ar) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:7 and a VL sequence having at least 95% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

as) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:7 and a VL sequence having at least 97% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

at) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:7 and a VL sequence having at least 99% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

ba) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bb a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bc) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bd) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

be) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bf) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bg) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bh) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bi) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bj) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO: and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bk) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bl) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bm) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bn) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bo) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bp) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:8 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

bq) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:8 and a VL sequence having at least 90% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

br) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:8 and a VL sequence having at least 95% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

bs) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:8 and a VL sequence having at least 97% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

bt) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:8 and a VL sequence having at least 99% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

ca) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cb a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cc) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cd) a VH sequence having at least 90% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ce) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cf) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cg) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ch) a VH sequence having at least 95% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ci) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cj) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

ck) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 99% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cl) a VH sequence having at least 97% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cm) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 90% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cn) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 95% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

co) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 97% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cp) a VH sequence having at least 99% identity to the sequence set forthin SEQ ID NO:9 and a VL sequence having at least 100% identity to thesequence set forth in SEQ ID NO:10, 11, or 12;

cq) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:9 and a VL sequence having at least 90% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

cr) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:9 and a VL sequence having at least 95% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12;

cs) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:9 and a VL sequence having at least 97% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12; and

ct) a VH sequence having at least 100% identity to the sequence setforth in SEQ ID NO:9 and a VL sequence having at least 99% identity tothe sequence set forth in SEQ ID NO:10, 11, or 12.

In one embodiment, the binding region comprises a VH and a VL selectedfrom the group consisting of;

a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as setforth in SEQ ID NO:10;

b) a VH sequence as set forth in SEQ ID NO:8, and a VL as set forth inSEQ ID NO:10;

c) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as setforth in SEQ ID NO:10;

d) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as setforth in SEQ ID NO:11;

e) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as setforth in SEQ ID NO:12;

f) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as setforth in SEQ ID NO:10;

g) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as setforth in SEQ ID NO:11;

h) a VH sequence as set forth in SEQ ID NO:7, and a VL sequence as setforth in SEQ ID NO:12;

i) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as setforth in SEQ ID NO:11;

j) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as setforth in SEQ ID NO:12;

k) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as setforth in SEQ ID NO:11; and

l) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as setforth in SEQ ID NO:12.

In a particular embodiment, the binding region comprises a VH sequenceand a VL sequence selected from the group consisting of;

a) a VH sequence as set forth in SEQ ID NO:6, and a VL sequence as setforth in SEQ ID NO:10;

b) a VH sequence as set forth in SEQ ID NO:8, and a VL sequence as setforth in SEQ ID NO:10; and

c) a VH sequence as set forth in SEQ ID NO:9, and a VL sequence as setforth in SEQ ID NO:10.

The humanized antibody according to the present invention may begenerated by comparison of the heavy and light chain variable regionamino acid sequences against a database of human germline variableregion sequences in order to identify the heavy and light chain humansequence with the appropriate degree of homology for use as humanvariable framework regions. A series of humanized heavy and light chainvariable regions may be designed by grafting, e.g. the murine, CDRs ontothe frameworks regions (identified as described above) and, ifnecessary, by back-mutation (mutation of one or more of the human aminoacid residues in the framework regions back to the non-human amino acidat the specific position(s)) to the specific murine sequence of residuesidentified which may be critical to the restoration of the antibodybinding efficiency. Variant sequences with the lowest incidence ofpotential T-cell epitopes may then be selected as determined byapplication of in silico technologies; iTope™ and TCED™ ([24], [25], and[26]).

Furthermore, the humanized antibodies according to the present inventionmay also be “deimmunized”. Deimmmunization may be desired, as within aprotein sequence, such as a humanized antibody according to the presentinvention, the presence of human T-cell epitopes may increase theimmunogenicity risk profile as they have the potential to activatehelper T-cells. Such activation of helper T-cells may be avoided bydeimmunization. Deimmunization may be performed by introducing amutation in the amino acid sequence of the humanized antibody in orderto remove the T-cell epitopes without significantly reducing the bindingaffinity of the antibody.

Thus, in one embodiment of the present invention, the humanized antibodymay be produced by a method comprising the steps of (i) comparing thenon-human full variable heavy chain sequence and/or the full variablelight chain sequence to a database of human germline sequences, (ii)selecting the human germline sequence having the highest homology to thenon-human sequence to obtain a humanized sequence, (iii) optimizing thehumanized sequence by back-mutation(s) if required, and (iv) expressingthe sequence in a suitable expression system.

Thus, a full-length antibody according to the present invention may beproduced by a method comprising the steps of (i) comparing the non-humanvariable heavy chain sequence and the variable light chain sequences toa database of human germline sequences, (ii) selecting the humangermline sequence having the highest homology to the non-human sequence,(iii) grafting of the non-human CDRs in to the selected human germ-lineto obtain a humanized sequences, (iv) optimizing the humanized sequencesby back-mutation(s) if required, (v) identifying constant heavy andlight chain sequences, and (vi) expressing the complete heavy chainsequences and complete light chain sequences in suitable expressionsystems. A full-length antibody according to the present invention may,thus, be produced as described in Example 1. It is within the knowledgeof the skilled person to produce a full-length antibody when startingout from either CDR sequences or full variable region sequences. Thus,the skilled person would know how to generate a full-length antibodyaccording to the present invention.

The term “complete heavy chain sequences” as used herein, refers to asequence consisting of variable heavy chain and constant heavy chainsequences.

The term “complete light chain sequences” as used herein, refers to asequence consisting of variable light chain and constant light chainsequences.

Back-mutation(s) may be introduced by standard DNA mutagenesis. Suchstandard techniques for DNA mutagenesis are described in [18].Alternatively, use of commercially available kits such as Quickchange™Site-Directed Mutagenesis Kit (Stratagene), or the desiredback-mutations may be introduced by de novo DNA synthesis.

Thus, in one embodiment, the antibody is a humanized antibody.

Chimeric antibodies may be generated by substituting all constant regionsequences of a non-human (such as murine) antibody with constant regionsequences of human origin. Thus, fully non-human variable regionsequences are maintained in the chimeric antibody. Thus, a chimericantibody according to the present invention may be produced by a methodcomprising the step of expressing the non-human variable heavy chain(SEQ ID NO:27), non-human variable light chain sequences (SEQ ID NO:28),human constant heavy chain and human constant light chain sequences insuitable expression systems, and thereby generating a full-lengthchimeric antibody. Alternative methods may be used. Such methods ofproducing a chimeric antibody is within the knowledge of the skilledperson, and thus, the skilled person would know how to produce achimeric antibody according to the present invention.

Thus, in one embodiment, the antibody is a chimeric antibody.

In one embodiment, the antibody is a full-length antibody. The term“full-length antibody” as used herein, refers to an antibody (e.g., aparent or variant antibody) which contains all heavy and light chainconstant and variable domains correspond to those that are normallyfound in a wild-type antibody of that isotype.

In one embodiment, the antibody comprises an Fc region comprising afirst and a second immunoglobulin heavy chain.

The term “Fc region” as used herein, refers to a region comprising, inthe direction from the N- to C-terminal, at least a hinge region, a CH2region and a CH3 region. An Fc region may further comprise a CH1 regionat the N-terminal end of the hinge region.

The term “hinge region” as used herein refers to the hinge region of animmunoglobulin heavy chain. Thus, for example the hinge region of ahuman IgG1 antibody corresponds to amino acids 216-230 according to theEu numbering as set forth in Kabat.

Unless otherwise stated or contradicted by context, the amino acids ofthe constant region sequences are herein numbered according to theEu-index of numbering (described in [27]) and may be termed “accordingto the Eu numbering as set forth in Kabat”, “Eu numbering according toKabat”, or “according to the Eu numbering system”.

The term “CH1 region” or “CH1 domain” as used herein, refers to the CH1region of an immunoglobulin heavy chain. Thus, for example the CH1region of a human IgG1 antibody corresponds to amino acids 118-215according to the Eu numbering system. However, the CH1 region may alsobe any of the other subtypes as described herein.

The term “CH2 region” or “CH2 domain” as used herein, refers to the CH2region of an immunoglobulin heavy chain. Thus, for example the CH2region of a human IgG1 antibody corresponds to amino acids 231-340according to the Eu numbering system. However, the CH2 region may alsobe any of the other subtypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein, refers to the CH3region of an immunoglobulin heavy chain. Thus, for example the CH3region of a human IgG1 antibody corresponds to amino acids 341-447according to the Eu numbering system. However, the CH3 region may alsobe any of the other subtypes as described herein.

In one embodiment, the isotype of the immunoglobulin heavy chain isselected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Theimmunoglobulin heavy chain may be any allotype within each of theimmunoglobulin classes, such as IgG1m(f) (SEQ ID NO:15). Thus, in oneparticular embodiment, the isotype of the immunoglobulin heavy chains isan IgG1, or any allotype thereof, such as IgG1m(f) (SEQ ID NO:15).

When targeting the antigen CD3 which is part of the T-cell Receptor(TCR), the T-cell specific mechanisms of cell killing is desirable.Other effector functions, e.g. complement activation, may not be wanted,and therefore, reduction of effector functions is desirable. C1q bindingis the first step in the complement cascade, and therefore serves as anindicator for complement-dependent cytotoxicity (CDC) capacity ofantibodies. If binding of C1q to the antibody can be avoided, activationof the complement cascade can be avoided as well.

Thus, in one embodiment, the antibody comprises an Fc region which hasbeen modified so that binding of C1q to said antibody is reducedcompared to a wild-type antibody by at least 70%, at least 80%, at least90%, at least 95%, at least 97%, at least 99%, or 100%, wherein C1qbinding is determined by ELISA.

The term “modified” as used herein, refers to the amino acid sequence ofan Fc region which is not identical to the amino acid sequence of awild-type Fc region. I.e. amino acid residues in specific positions ofthe wild-type Fc region have been substituted, deleted or inserted inorder to alter, for example, the binding site for C1q, binding site forother effector molecules or binding to Fc Receptors (FcRs). Suchmodification(s) of the amino acid sequence may be prepared bysubstituting one or more amino acids with a conservative amino acid ormay be prepared by substituting one or more amino acids with analternative amino acid which is physically and/or functionally similarto the amino acid present in the wild-type. Substitutions may also beprepared by substituting with a non-conservative amino acid.

In the context of the present invention, amino acids may be described asconservative or non-conservative amino acids, and may therefore beclassified accordingly. Amino acid residues may also be divided intoclasses defined by alternative physical and functional properties. Thus,classes of amino acids may be reflected in one or both of the followingtables:

Amino Acid Residue of Conservative Class

Acidic Residues D and E Basic Residues K, R, and H Hydrophilic UnchargedResidues S, T, N, and Q Aliphatic Uncharged Residues G, A, V, L, and INon-polar Uncharged Residues C, M, and P Aromatic Residues F, Y, and W

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turn A,C, D, E, G, H, K, N, Q, R, S, P, formation and T Flexible residues Q, T,K, S, G, P, D, E, and R

In the context of the present invention, a substitution in an antibody,such as a humanized or chimeric antibody, is indicated as:

Original amino acid—position—substituted amino acid;

Referring to the well-recognized nomenclature for amino acids, the threeletter code, or one letter code, is used, including the codes Xaa and Xto indicate any amino acid residue. Accordingly, the notation “L234F” or“Leu234Phe” means, that the antibody comprises a substitution of Leucinewith Phenylalanine in amino acid position 234.

Substitution of an amino acid at a given position to any other aminoacid is referred to as:

Original amino acid—position; or e.g. “L234”.

For a modification where the original amino acid(s) and/or substitutedamino acid(s) may comprise more than one, but not all amino acid(s), themore than one amino acid may be separated by “,” or “/”. E.g. thesubstitution of Leucine for Phenylalanine, Arginine, Lysine orTryptophan in position 234 is:

“Leu234Phe,Arg,Lys,Trp” or “Leu234Phe/Arg/Lys/Trp” or “L234F,R,K,W” or“L234F/R/K/W” or “L234 to F, R, K or W”

Such designation may be used interchangeably in the context of theinvention but have the same meaning and purpose.

Furthermore, the term “a substitution” embraces a substitution into anyone of the other nineteen natural amino acids, or into other aminoacids, such as non-natural amino acids. For example, a substitution ofamino acid L in position 234 includes each of the followingsubstitutions: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K,234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. This is,by the way, equivalent to the designation 234X, wherein the X designatesany amino acid other than the original amino acid. These substitutionscan also be designated L234A, L234C, etc., or L234A,C, etc., orL234A/C/etc. The same applies by analogy to each and every positionmentioned herein, to specifically include herein any one of suchsubstitutions.

The antibody according to the invention may also comprise a deletion ofan amino acid residue. Such deletion may be denoted “del”, and includes,e.g., writing as L234del. Thus, in such embodiments, the Leucine inposition 234 has been deleted from the amino acid sequence.

The terms “amino acid” and “amino acid residue” may herein be usedinterchangeably.

The term “C1q binding” as used herein, refers to the binding of C1q toan antibody, when said antibody is bound to its antigen. The term “boundto its antigen” as used herein, refers to binding of an antibody to itsantigen both in vivo and in vitro.

The term “reduce” as used herein when referring to C1q binding, refersto the ability of the antibody according to the invention to reduce,minimize or even completely inhibit the binding of C1q to the antibodywhen compared to the C1q binding to a wild-type antibody.

The term “wild-type antibody” as used herein, in relation to use incomparison assays of an antibody according to the present invention,refers to an antibody which is identical to the antibody to be testedexcept for not being inert. In this context, the term “inert” refers toa modified Fc region having reduced or no binding of C1q as determinedin Example 10, i.e. where C1q binding is determined by ELISA; reduced orno Fc-mediated T-cell proliferation as determined in Example 4, i.e.T-cell proliferation is measured in a peripheral blood mononuclear cell(PBMC)-based functional assay; and/or reduced or no Fc-mediated CD69expression as determined in Example 3, i.e. Fc-mediated CD69 expressionis determined in a PBMC-based functional assay. Thus, the wild-typeantibody comprises the naturally occurring amino acids in theimmunoglobulin heavy chains, i.e. an antibody which does not compriseany amino acid modifications which may alter or reduce the ability ofthe antibody to interact with e.g. C1q, Fc Receptors or the like. Thus,such a wild-type antibody will remain an activating antibody which isable to bind e.g. C1q. A wild-type antibody and an antibody of thepresent invention may comprise other amino acid modifications than thoseaffecting the antibody's ability of inducing effector functions, inorder to make the antibody a bispecific antibody or the like.

The term “ELISA” as used herein refers to enzyme-linked immunosorbentassay which is a test that uses antibodies and color change to identifya substance. A first specific antibody is attached to the plate surface.Thereby the protein from a sample is added wherein binding to said firstspecific antibody is tested. A second antibody binding the antibody fromthe sample is added. The second antibody is linked to an enzyme, and, inthe final step, a substance containing the enzyme's substrate is added.The subsequent reaction produces a detectable signal, most commonly acolor change in the substrate. The concept of the ELISA method iswell-known within the art and various ways of performing an ELISA arecontemplated to be part of a method to evaluate the antibody accordingto the invention. Thus, this interpretation is not to be understood aslimiting as various forms of ELISAs may be performed such as describedin Example 4.

Specifically, the ability of an antibody according to the presentinvention to bind C1q may be determined by ELISA comprising the steps of(i) coating said antibody on a 96-well plate, (ii) adding 3% serum,(iii) adding an anti-human C1q antibody, (iv) developing the plate, and(v) measuring OD₄₀₅ nm. Thus, in one embodiment, the antibody comprisesan Fc region which has been modified so that binding of C1q to saidantibody is reduced compared to a wild-type antibody by at least 70%, atleast 80%, at least 90%, at least 95%, at least 97%, or 100%, whereinC1q binding is determined by ELISA comprising the steps of (i) coatingsaid antibodies on a 96-well plate, (ii) adding 3% serum, (iii) addingan anti-human C1q, (iv) developing the plate, and (v) measuring OD₄₀₅nm. Thus, in particular embodiment, binding of C1q is evaluated asdescribed in Example 10.

The terms “Fc Receptor” or “FcR” as used herein, refers to a proteinfound on the surface of certain cells. FcRs bind to the Fc region ofantibodies. There are several different types of FcRs which areclassified based on the type of antibody they recognize. E.g. Fcγ(gamma) Receptors bind to antibodies of the IgG class.

The terms “Fcγ Receptor”, “Fc gamma Receptor” or “FcγR” as used herein,refers to a group of Fc Receptors belonging to the immunoglobulinsuperfamily and is the most important Fc receptors for inducingphagocytosis of opsonized (coated) microbes. This family includesseveral members, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b),FcγRIIIa (CD16a), FcγRIIIb (CD16b), which differ in their antibodyaffinities due to their different molecular structure.

Fc-mediated effector functions form part of the biological activity ofhuman immunoglobulin G (IgG) molecules. Examples of such effectorfunctions include e.g. antibody-dependent cell-mediated cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) which are triggeredby the binding of various effector molecules to the Fc region. In thecontext of the present invention, “Fc binding”, “Fc Receptor binding”,“FcR binding”, and “binding of an antibody Fc region to FcR” refers tothe binding of the Fc region to an Fc Receptor (FcR) or an effectormolecule. The terms “FcγR binding” and “FcγRI binding” refer to bindingto or with an Fc region to the Fc gamma Receptor and Fc gamma ReceptorI, respectively. When a CD3 antibody binds T-cells, the wild-type Fcregion of the CD3 antibody binds to FcRs present on other cells, e.g.monocytes, which leads to non-specific, Fc-mediated activation of theT-cell. Such non-specific, Fc-mediated activation of T-cells may beundesired. T-cells may also be activated by targeted, ortarget-specific, T-cell activation. Such targeted T-cell activation maybe highly desirable for the treatment of a range of indications, such ascancer. The term “targeted T-cell activation” as used herein, refers todirecting the T-cells to specific cells, such as tumor cells by use of abispecific antibody comprising a first binding region binding a specifictarget, such as a tumor target on a tumor cell, and a second bindingregion binding a T-cell specific target, such as CD3. Thus, targeting ofT-cells to specific cells, e.g. tumor cells, may be facilitated by useof a bispecific antibody, wherein one of the binding regions binds CD3present on the T-cell and the other binding region binds a targetspecific antigen, e.g. on a tumor cell. Although, non-specific,Fc-mediated T-cells activation may still be possible and therefore suchundesired non-specific, Fc-mediated T-cell activation via Fc-mediatedcross-linking should be avoided and may be disabled by making the Fcregion inert for such activity. Thereby, interaction between said inertFc region with Fc Receptors present is prevented. A humanized antibodyof the present invention has been proven to be inert when tested inseveral different assays, i.e. see Examples 3 to 5. Another tested CD3antibody, huCLB-T3/4, comprising amino acid modifications in the Fcregion also proved to be inert when tested in different assays, i.e. seeExamples 7 to 10. The humanized CD3 antibody according to the presentinvention comprising the amino acid substitutions L234F, L235E, andD265A, as described in the Examples, showed low levels of CD69expression on T-cells (Example 3), abrogation of Fc-mediated T-cellproliferation (Example 4), and no non-specific target killing when inthe form of a bispecific antibody (Example 5). Thus, a humanizedantibody of the present invention shows superior results in severalassays when compared to a wild-type antibody.

An antibody according to the present invention may comprisemodifications in the Fc region. When an antibody comprises suchmodifications it may become an inert, or non-activating, antibody. Theterm “inertness”, “inert” or “non-activating” as used herein, refers toan Fc region which is at least not able to bind any Fcγ Receptors,induce Fc-mediated cross-linking of FcRs, or induce FcR-mediatedcross-linking of target antigens via two Fc regions of individualantibodies, or is not able to bind C1q. The inertness of an Fc region ofa humanized or chimeric CD3 antibody is advantageously tested using theantibody in a monospecific format although an inert Fc region soidentified can be used in bispecific or other humanized or chimericmultispecific CD3 antibodies.

Several variants can be constructed to make the Fc region of an antibodyinactive for interactions with Fc gamma Receptors and C1q fortherapeutic antibody development. Examples of such variants aredescribed herein.

Thus, in one embodiment, the antibody comprises an Fc region which hasbeen modified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

The term “reduce” as used herein, refers to a reduction of activity orexpression when compared to a control protein, such as an antibody. Inparticular, the term “reduce” when referring to T-cell proliferation,refers to the ability of the antibody according to the invention toreduce, minimize or even completely inhibit the proliferation of T-cellswhen compared to the proliferation of T-cells bound by a wild-typeantibody. The ability of an antibody to reduce T-cell proliferation maybe evaluated by a PBMC-based functional assay, as described in Example 4and Example 8. In one embodiment the assay is performed with humanPBMCs. In another embodiment the assay is performed with cynomolgusPBMCs. In yet another embodiment, the assay is performed with rhesusPBMCs. Since the antibodies according to the present invention arecross-reactive, a PBMC-based assay as herein described may be performedwith any species PBMCs to show reduction of T-cell proliferation as longas the species PBMC used are within the cross-reactivity spectra of theantibodies, e.g. human, cynomolgus or rhesus monkeys.

The term “peripheral blood mononuclear cell (PBMC)-based functionalassay” as used herein refers to an assay used for evaluating afunctional feature of the antibody of the present invention, such as theability of said antibody to affect T-cell proliferation or CD69expression, wherein the only cells present are peripheral bloodmononuclear cells. Thus, in one embodiment, T-cell proliferation ismeasured by a method comprising the steps of incubating PBMCs withantibody in the range of 1-1000 ng/mL at 37° C. in a 5% (vol/vol) CO₂humidified incubator for three days, adding a chemical compound, such asBrdU, which is incorporated into the DNA of proliferating cells,incubating for five hrs., pelleting cells, drying cells, optionallystoring the cells at 4° C., coating cells to ELISA plates, incubatingwith anti-BrdU-peroxidase for 90 min at room temperature, developing forabout 30 min with 1 mg/mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), adding 100 μL 2% oxalic acidto stop the reaction, and measuring absorbance at 405 nm in a suitablemicroplate reader.

The term “proliferation” as used herein, refers to cell growth in thecontext of cell division.

The term “BrdU” as used herein, refers to 5-bromo-2′-deoxyuridine, whichis a homologue to thymidine. When BrdU is added to cell culture for alimited period of time (e.g. 4 hours) it will be incorporated into theDNA of proliferating cells. After fixing the cells, detection ofincorporated BrdU may be performed in an ELISA usinganti-BrdU-peroxidase. BrdU incorporation is therefore a measure forproliferation.

In one embodiment, the antibody comprises an Fc region which has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

The term “reduce” as used herein, refers to a reduction of activity orexpression when compared to a control protein, such as an antibody. Inparticular, the term “reduce” when referring to expression level of theT-cell activation marker CD69, refers to a reduction in expression levelof CD69 when compared to expression level of CD69 when the T-cell isbound by a wild-type antibody provided that both the binding regions ofthe antibody binds CD3. An antibody's ability to reduce expression ofCD69 may be evaluated by a PBMC-based functional assay, as described inExample 3 and Example 7. Thus, in one embodiment, expression of CD69 ismeasured by a method comprising the steps of incubating PBMCs with anantibody in the range of 1-1000 ng/mL at 37° C. in a 5% (vol/vol) CO₂humidified incubator for 16-24 hrs, washing the cells, staining thecells at 4° C. with a mouse anti-human CD28-PE and mouse-anti-humanCD69-APC antibody, and determining CD69-expression on CD28 positivecells by flow cytometry.

The term “CD69” as used herein, refers to Cluster of Differentiation 69which is a human transmembrane C-Type lectin protein encoded by the CD69gene. Activation of T lymphocytes and natural killer (NK) cells, both invivo and in vitro, induces expression of CD69. CD69 function as a signaltransmitting receptor involved in cellular activation events includingproliferation, functions as a signal-transmitting receptor inlymphocytes, including natural killer cells and platelets, and theinduction of specific genes.

The term “peripheral blood mononuclear cell (PBMC)-based functionalassay” as used herein refers to an assay used for evaluating afunctional feature of the antibody of the present invention, such as theability of said antibody to affect T-cell proliferation or CD69expression, wherein the only cells present are peripheral bloodmononuclear cells. A PBMC-based functional assay as described in Example3, 4, 5, and 7 comprises the steps of (i) incubating PBMCs with anantibody at 37° C. in a 5% (vol/vol) CO₂ humidified incubator for about16-24 hrs, (ii) washing the cells, (iii) staining the cells at 4° C.with a mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibody,and (iv) determining the CD69 expression on CD28 positive cells by flowcytometry, when CD69 expression is evaluated. Thus, in one embodiment,CD69 expression may be determined as described in Example 3, 4, 5, or 7.

Thus, amino acids in the Fc region that play a dominant role in theinteractions with C1q and the Fc Gamma Receptors may be modified.Examples of amino acid positions that may be modified include positionsL234, L235 and P331. Combinations thereof, such as L234F/L235E/P331S,can cause a profound decrease in binding to human CD64, CD32A, CD16 andC1q.

Hence, in one embodiment, the amino acid in at least one positioncorresponding to L234, L235 and P331, may be A, A and S, respectively([1], [28]). Also, L234F and L235E amino acid substitutions can resultin Fc regions with abrogated interactions with Fc Gamma Receptors andC1q ([29]-[30]). Hence, in one embodiment, the amino acids in thepositions corresponding to L234 and L235, may be F and E, respectively.A D265A amino acid substitution can decrease binding to all Fc gammaReceptors and prevent ADCC ([31]). Hence, in one embodiment, the aminoacid in the position corresponding to D265 may be A. Binding to C1q canbe abrogated by mutating positions D270, K322, P329, and P331. Mutatingthese positions to either D270A or K322A or P329A or P331A can make theantibody deficient in CDC activity ([32]). Hence, in one embodiment, theamino acids in at least one position corresponding to D270, K322, P329and P331, may be A, A, A, and A, respectively.

An alternative approach to minimize the interaction of the Fc regionwith Fc gamma Receptors and C1q is by removal of the glycosylation siteof an antibody. Mutating position N297 to e.g. Q, A, and E removes aglycosylation site which is critical for IgG-Fc gamma Receptorinteractions. Hence, in one embodiment, the amino acid in a positioncorresponding to N297, may be G, Q, A or E ([33]). Another alternativeapproach to minimize interaction of the Fc region with Fc gammaReceptors may be obtained by the following mutations; P238A, A327Q,P329A or E233P/L234V/L235A/G236del ([31]).

Alternatively, human IgG2 and IgG4 subclasses are considered naturallycompromised in their interactions with C1q and Fc gamma Receptorsalthough, interactions with Fcγ Receptors (Fc gamma Receptors) werereported ([34]-[35]). Mutations abrogating these residual interactionscan be made in both isotypes, resulting in reduction of unwantedside-effects associated with FcR binding. For IgG2, these include L234Aand G237A, and for IgG4, L235E. Hence, in one embodiment, the amino acidin a position corresponding to L234 and G237 in a human IgG2 heavychain, may be A and A, respectively. In one embodiment, the amino acidin a position corresponding to L235 in a human IgG4 heavy chain, may beE.

Other approaches to further minimize the interaction with Fc gammaReceptors and C1q in IgG2 antibodies include those described in [36] and[37].

The hinge region of the antibody can also be of importance with respectto interactions with Fc gamma Receptors and complement ([38]-[39]).Accordingly, mutations in or deletion of the hinge region can influenceeffector functions of an antibody.

The term “cross-linking” as used herein, refers to the indirect bridgingof antibody Fab arm(s) (monovalently or bivalently) bound to the targetantigen by FcR-bearing cell through binding to the antibody Fc region.Thus, an antibody which binds its target antigen on targetantigen-bearing cells may cross-link with another cell expressing FcRs.

The term “unspecific killing” as used herein, refers to the killing ofcells by the cytotoxic function of T-cells or other effector cells,through tumor target antigen-independent activation of said cells. Thus,by unspecific killing is meant that the tumor-target bearing cells maybe killed by e.g. cytotoxic T-cells and not by the antibody binding thetumor target by e.g. induction of CDC.

The present inventors have shown (see Examples 3 to 5, 7 to 10) that anon-activating Fc region may be obtained by modifying one or more of atleast five specific amino acid positions in the Fc region.

Thus, in one embodiment, the antibody comprises a first and a secondimmunoglobulin heavy chain, wherein in at least one of said first andsecond immunoglobulin heavy chains one or more amino acids in thepositions corresponding to positions L234, L235, D265, N297, and P331 ina human IgG1 heavy chain, are not L, L, D, N, and P, respectively.

In one embodiment, in both the first and second heavy chains one or moreamino acids in the position corresponding to positions L234, L235, D265,N297, and P331 in a human IgG1 heavy chain, are not L, L, D, N, and P,respectively.

In another embodiment, in at least one of the first and second heavychains one or more amino acids in the positions corresponding topositions L234, L235 and D265 in a human IgG1 heavy chain, are not L, Land D, respectively, and the amino acids in the positions correspondingto N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

The term “amino acid corresponding to positions” as used herein refersto an amino acid position number in a human IgG1 heavy chain. Unlessotherwise stated or contradicted by context, the amino acids of theconstant region sequences are herein numbered according to the Eu-indexof numbering (described in [27]). Thus, an amino acid or segment in onesequence that “corresponds to” an amino acid or segment in anothersequence is one that aligns with the other amino acid or segment using astandard sequence alignment program such as ALIGN, ClustalW or similar,typically at default settings and has at least 50%, at least 80%, atleast 90%, or at least 95% identity to a human IgG1 heavy chain. It isconsidered well-known in the art how to align a sequence or segment in asequence and thereby determine the corresponding position in a sequenceto an amino acid position according to the present invention.

In the context of the present invention, the amino acid may be definedas described above.

The term “the amino acid is not” or similar wording when referring toamino acids in a heavy chain is to be understood to mean that the aminoacid is any other amino acid than the specific amino acid mentioned. Forexample, the amino acid in the position corresponding to L234 in a humanIgG1 heavy chain is not L, means that the amino acid may be any of theother naturally or non-naturally occurring amino acids than L.

In one embodiment, in at least one of said first and second heavy chainsthe amino acid in the position corresponding to position D265 in a humanIgG1 heavy chain, is not D.

In one embodiment, in at least one of the first and second heavy chainsthe amino acid in the position corresponding to D265 in a human IgG1heavy chain, is not D, and the amino acids in the positionscorresponding to positions N297 and P331 in a human IgG1 heavy chain,are N and P, respectively.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to position D265 in ahuman IgG1 heavy chain is hydrophobic or polar amino acids.

The term “hydrophobic” as used herein in relation to an amino acidresidue, refers to an amino acid residue selected from the groupconsisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in oneembodiment, in at least one of said first and second heavy chains theamino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group of amino acids consistingof; A, C, F, G, H, I, L, M, R, T, V, W and Y.

The term “polar” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of;C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acid in the positioncorresponding to position D265 in a human heavy chain is selected fromthe group consisting of; C, E, H, K, N, Q, R, S, and T.

In another embodiment, in at least one of said first and second heavychains the amino acid in the position corresponding to position D265 ina human IgG1 heavy chain is an aliphatic uncharged, aromatic or acidicamino acid.

The term “aliphatic uncharged” as used herein in relation to amino acidresidues, refers to any amino acid residue selected from the groupconsisting of: A, G, I, L, and V. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acid in the positioncorresponding to position D265 in a human IgG1 heavy chain is selectedfrom the group consisting of; A, G, I, L, and V.

The term “aromatic” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of:F, T, and W. Thus, in one embodiment, in at least one of said first andsecond heavy chains the amino acid in the position corresponding toposition D265 in a human IgG1 heavy chain is selected from the groupconsisting of; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues,refers to any amino acid residue chosen from the group consisting of: Dand E. Thus, in one embodiment, in at least one of said first and secondheavy chains the amino acid in the position corresponding to positionD265 in a human IgG1 heavy chain is selected from the group consistingof; D and E.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acid in the position corresponding to positionD265 in a human IgG1 heavy chain is selected from the group consistingof; A, E, F, G, I, L, T, V, and W.

In one embodiment, in both said first and second heavy chains the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain, is not D.

In one embodiment, in both the first and second heavy chains the aminoacid in the position corresponding to D265 in a human IgG1 heavy chain,is not D, and the amino acids in the positions corresponding topositions N297 and P331 in a human IgG1 heavy chain, are N and P,respectively.

In one embodiment, in both said first and second heavy chains the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain is hydrophobic or polar amino acid.

The term “hydrophobic” as used herein in relation to an amino acidresidue, refers to an amino acid residue selected from the groupconsisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in oneembodiment, in both said first and second heavy chains the amino acid inthe position corresponding to position D265 in a human IgG1 heavy chainis selected from the group of amino acids consisting of; A, C, F, G, H,I, L, M, R, T, V, W and Y.

The term “polar” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of;C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in both saidfirst and second heavy chains the amino acid in the positioncorresponding to position D265 in a human heavy chain is selected fromthe group consisting of; C, E, H, K, N, Q, R, S, and T. In oneembodiment, in both said first and second heavy chains the amino acid inthe position corresponding to position D265 in a human IgG1 heavy chainis selected from the group of amino acids consisting of; A, C, F, G, H,I, L, M, R, T, V, W and Y.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to position D265 in a human heavychain is selected from the group consisting of; C, E, H, K, N, Q, R, S,and T.

In another embodiment, in both said first and second heavy chains theamino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is aliphatic uncharged, aromatic or acidic amino acids.

The term “aliphatic uncharged” as used herein in relation to amino acidresidues, refers to any amino acid residue selected from the groupconsisting of: A, G, I, L, and V. Thus, in one embodiment, in both saidfirst and second heavy chains the amino acid in the positioncorresponding to position D265 in a human IgG1 heavy chain is selectedfrom the group consisting of; A, G, I, L, and V.

The term “aromatic” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of:F, T, and W. Thus, in one embodiment, in both said first and secondheavy chains the amino acid in the position corresponding to positionD265 in a human IgG1 heavy chain is selected from the group consistingof; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues,refers to any amino acid residue chosen from the group consisting of: Dand E. Thus, in one embodiment, in both said first and second heavychains the amino acid in the position corresponding to position D265 ina human IgG1 heavy chain are selected from the group consisting of; Dand E.

In a particular embodiment, in both said first and second heavy chainsthe amino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group consisting of; A, E, F, G,I, L, T, V, and W.

In further embodiment, in at least one of said first and second heavychains the amino acid in the position corresponding to position N297 ina human IgG1 heavy chain, is not N.

In one embodiment, in at least one of the first and second heavy chainsthe amino acid in the position corresponding to N297 in a human IgG1heavy chain, is not N, and the amino acid in the position correspondingto position P331 in a human IgG1 heavy chain, is P.

In one embodiment, in both said first and second heavy chains the aminoacid in the position corresponding to positions N297 in a human IgG1heavy chain, is not N.

In one embodiment, in both the first and second heavy chains the aminoacid in the position corresponding to N297 in a human IgG1 heavy chain,is not N, and the amino acid in the position corresponding to positionP331 in a human IgG1 heavy chain, is P.

In further embodiment, in at least one of said first and second heavychains the amino acids in the positions corresponding to positions L234and L235 in a human IgG1 heavy chain, are not L and L, respectively.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234 and L235 in ahuman IgG1 heavy chain, are not L and L, respectively, and the aminoacids in the positions corresponding to positions N297 and P331 in ahuman IgG1 heavy chain, are N and P, respectively.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids corresponding to positions L234 and L235 in a human IgG1heavy chain are selected from the group consisting of; A, C, D, E, F, G,H, I, K, M, N, P, Q, R, S, T, Y, V.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

The term “hydrophobic” as used herein in relation to an amino acidresidue, refers to an amino acid residue selected from the groupconsisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in oneembodiment, in at least one of said first and second heavy chains theamino acids in the positions corresponding to positions L234 and L235 ina human IgG1 heavy chain are each selected from the group consisting of;A, C, F, G, H, I, M, R, T, V, W, and Y.

The term “polar” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of;C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group of amino acids consisting of; C,D, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain are each selected from thegroup consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W,and Y.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain, are not L and L, respectively.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234 and L235 in a human IgG1heavy chain, are not L and L, respectively, and the amino acids in thepositions corresponding to positions N297 and P331 in a human IgG1 heavychain, are N and P, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to L234 and L235 in a human IgG1heavy chain are hydrophobic or polar amino acids.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain are each selected from the group consisting of;A, C, F, G, H, I, M, R, T, V, W, and Y.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain are each selected from the group of amino acidsconsisting of; C, D, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in both said first and second heavy chainsthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are each selected from the groupconsisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

In another embodiment, in at least one of said first and second heavychains the amino acids in the positions corresponding to positions L234and L235 in a human IgG1 heavy chain are aliphatic uncharged, aromaticor acidic amino acids.

The term “aliphatic uncharged” as used herein in relation to amino acidresidues, refers to any amino acid residue selected from the groupconsisting of: A, G, I, L, and V. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group consisting of; A, G, I, and V.

The term “aromatic” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of:F, T, and W. Thus, in one embodiment, in at least one of said first andsecond heavy chains the amino acids in the positions corresponding topositions L234 and L235 in a human IgG1 heavy chain are each selectedfrom the group consisting of; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues,refers to any amino acid residue chosen from the group consisting of: Dand E. Thus, in one embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain are each selected from thegroup consisting of; D and E.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to L234 andL235 are each selected from the group consisting of; A, D, E, F, G, I,T, V, and W.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234 and L235 in ahuman IgG1 heavy chain, are F and E; or A and A, respectively, and theamino acids in the positions corresponding to positions N297 and P331 ina human IgG1 heavy chain, are N and P, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain, are F and E; or A and A, respectively.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234 and L235 in a human IgG1heavy chain, are F and E; or A and A, respectively, and the amino acidsin the positions corresponding to positions N297 and P331 in a humanIgG1 heavy chain, are N and P, respectively.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain, are F and E, respectively.

In one embodiment, in at least one of said first and second heavy chainsat least the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

In one embodiment, in both said first and second heavy chains at leastthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain, are A and A, respectively.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234, L235,and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234, L235, and D265in a human IgG1 heavy chain, are not L, L and D, respectively, and theamino acids in the positions corresponding to positions N297 and P331 ina human IgG1 heavy chain, are N and P, respectively.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids corresponding to positions L234 and L235 in a human IgG1heavy chain are selected from the group consisting of; A, C, D, E, F, G,H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acidcorresponding to position D265 is selected from the group consisting of;A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234, L235and D265 in a human IgG1 heavy chain are hydrophobic or polar aminoacids.

The term “hydrophobic” as used herein in relation to an amino acidresidue, refers to an amino acid residue selected from the groupconsisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in oneembodiment, in at least one of said first and second heavy chains theamino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group of amino acids consistingof; A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group consisting of; A, C, F, G, H, I,M, R, T, V, W, and Y.

The term “polar” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of;C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group of amino acids consisting of; C,D, E, H, K, N, Q, R, S, and T, the amino acid in the positioncorresponding to position D265 in a human heavy chain is selected fromthe group consisting of; C, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain are each selected from thegroup consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W,and Y, and the amino acid in the position corresponding to position D265in a human IgG1 heavy chain is selected from the group consisting of; A,C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain are hydrophobic or polar amino acids.

In one embodiment, in both said first and second heavy chains the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain is selected from the group of amino acids consisting of; A,C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group consisting of; A, C, F, G, H, I,M, R, T, V, W, and Y.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain are each selected from the group of amino acidsconsisting of; C, D, E, H, K, N, Q, R, S, and T, the amino acid in theposition corresponding to position D265 in a human heavy chain isselected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in both said first and second heavy chainsthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are each selected from the groupconsisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y,and the amino acid in the position corresponding to position D265 in ahuman IgG1 heavy chain is selected from the group consisting of; A, C,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In another embodiment, in at least one of said first and second heavychains the amino acids in the positions corresponding to positions L234,L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged,aromatic or acidic amino acids.

The term “aliphatic uncharged” as used herein in relation to amino acidresidues, refers to any amino acid residue selected from the groupconsisting of: A, G, I, L, and V. Thus, in one embodiment, in at leastone of said first and second heavy chains the amino acid in the positioncorresponding to position D265 in a human IgG1 heavy chain is selectedfrom the group consisting of; A, G, I, L, and V, and the amino acids inthe positions corresponding to positions L234 and L235 in a human IgG1heavy chain are each selected from the group consisting of; A, G, I, andV.

The term “aromatic” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of:F, T, and W. Thus, in one embodiment, in at least one of said first andsecond heavy chains the amino acids in the positions corresponding topositions L234, L235 and D265 in a human IgG1 heavy chain are eachselected from the group consisting of; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues,refers to any amino acid residue chosen from the group consisting of: Dand E. Thus, in one embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain are each selected fromthe group consisting of; D and E.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acid in the position corresponding to positionD265 in a human IgG1 heavy chain is selected from the group consistingof; A, E, F, G, I, L, T, V, and W, and the amino acids in the positionscorresponding to L234 and L235 are each selected from the groupconsisting of; A, D, E, F, G, I, T, V, and W.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235 and D265 ina human IgG1 heavy chain, are not L, L, and D, respectively.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain, are not L, L, and D, respectively, and the amino acidsin the positions corresponding to positions N297 and P331 in a humanIgG1 heavy chain, are N and P, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain are aliphatic uncharged, aromatic or acidic aminoacids.

In one embodiment, in both said first and second heavy chains the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain is selected from the group consisting of; A, G, I, L, and V,and the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are each selected from the groupconsisting of; A, G, I, and V.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain are each selected from the group consistingof; D and E.

In a particular embodiment, in both said first and second heavy chainsthe amino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group consisting of; A, E, F, G,I, L, T, V, and W, and the amino acids in the positions corresponding toL234 and L235 are each selected from the group consisting of; A, D, E,F, G, I, T, V, and W.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234, L235,and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A,respectively.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234, L235, and D265in a human IgG1 heavy chain, are F, E, and A; or A, A, and A,respectively, and the amino acids in the positions corresponding topositions N297 and P331 in a human IgG1 heavy chain, are N and P,respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain, are F, E, and A; or A, A, and A,respectively.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain, are F, E, and A; or A, A, and A, respectively, and theamino acids in the positions corresponding to positions N297 and P331 ina human IgG1 heavy chain, are N and P, respectively.

In a particular embodiment, in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain, are F, E, and A, respectively.

In one embodiment, in at least one of said first and second heavy chainsthe amino acids in the positions corresponding to positions L234, L235,and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain, are A, A, and A, respectively.

In another embodiment, in at least one of said first and second heavychains the amino acids in the positions corresponding to positions L234,L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q,and S, respectively.

In one embodiment, in both said first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, D265,N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S,respectively.

In a particular embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:8, a VL sequence as setout in SEQ ID NO:10, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In another embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:8, a VL sequence as setout in SEQ ID NO:12, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In another embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:6, a VL sequence as setout in SEQ ID NO:10, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In another embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:6, a VL sequence as setout in SEQ ID NO:12, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In another embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:9, a VL sequence as setout in SEQ ID NO:10, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In another embodiment, the antibody according to the invention,comprises a VH sequence as set out in SEQ ID NO:9, a VL sequence as setout in SEQ ID NO:12, and in at least one of the heavy chains the aminoacids in positions corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain, are F, E, and A, respectively.

In one aspect, the antibody according to the invention comprises thehuman IgLC2/IgLC3 constant domain lambda light chain of SEQ ID NO:31.

In one aspect, the antibodies according to the invention may be modifiedin the light chain (LC) and/or heavy chain (HC) to increase theexpression level and/or production yield. In one embodiment, theantibodies according to the invention may be modified in the light chain(LC). Such modifications are known in the art and may be performedaccording to the methods described in e.g. Zheng, L., Goddard, J.-P.,Baumann, U., & Reymond, J.-L. (2004). Expression improvement andmechanistic study of the retro-Diels-Alderase catalytic antibody 10F11by site-directed mutagenesis. Journal of Molecular Biology, 341(3),807-14. doi:10.1016/j.jmb.2004.06.014.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position T41 in the lambda light chain of SEQ ID NO:10 is not T.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position T41 in the lambda light chain of SEQ ID NO:10 is selectedfrom H, I, K, L, Q, R and V.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position T41 in the lambda light chain of SEQ ID NO:10 is H, K or R,such as K.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position F10 in the lambda light chain of SEQ ID NO:10 is not F, andwherein one or more of the amino acid positions corresponding to thepositions T41, K55, and L97 in the lambda light chain of SEQ ID NO:10are not T, K and L, respectively.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acids in the positions correspondingto positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO:10 are not F, T, K and L, respectively.

In one embodiment the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position T41 is selected from H, I, K, L, Q, R or V, such as selectedfrom H, K and R, such as K, and wherein the amino acids in the positionscorresponding to positions F10, T41, K55, and L97 in the lambda lightchain of SEQ ID NO:10 are L, K, N, and H, respectively.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acids in the positions correspondingto positions R23 and A35 are not R and A, respectively.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position R23 is selected from A, G, H, K, Q, S, and T, such as from Aand G, and wherein the amino acid in the position corresponding to A35is selected from I, L, M, P, V, G, F and W, such as from I, L, M, P, andV.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position R23 is A or G, such as A, and the amino acid in the positioncorresponding to position A35 is P.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acids in the positions correspondingto positions F10, R23, A35, R47, D71, A82, D83, S86, 187, and F89 in thelambda light chain of SEQ ID NO:10 are not F, R, A, R, D, A, D, S, I,and F, respectively.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position R23 is selected from A, G, H, K, Q, S, and T, such as from Aand G, wherein the amino acid in the position corresponding to A35 isselected from I, L, M, P, V, G, F and W, such as from I, L, M, P, andwherein the amino acids in the positions corresponding to positions F10,R47, D71, A82, D83, S86, 187, and F89 in the lambda light chain of SEQID NO:10 are L, T, G, P, E, A, E, and Y, respectively.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acid in the position correspondingto position R23 is A or G, and wherein the amino acids in the positionscorresponding to positions F10, A35, R47, D71, A82, D83, S86, 187, andF89 in the lambda light chain of SEQ ID NO:10 are L, P, T, G, P, E, A,E, and Y, respectively.

In one aspect, the antibodies according to the invention may be modifiedin the light chain (LC) to increase the affinity of the antibodies.Modifications that lead to increased antibody affinity are known in theart.

In one aspect, the antibodies according to the invention may be modifiedin the light chain (LC) to reduce the affinity of the antibodies. Thismay be advantageous in some settings and lead to increased efficacy. Inparticular low affinity of the CD3 arm may have an impact on themotility of T cells in circulation and at tumor site thus leading tobetter engagement of T cells with tumor cells, cf. Mølhøj et al,Molecular Immunology 44 (2007). In particular this may be useful inbispecific formats, in which the CD3 antibodies are used as one of thebinding arms. Modifications that lead to reduced antibody affinity areknown in the art, see for example Webster et al. Int J Cancer Suppl.1988; 3:13-6.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein

(i) the amino acid in the position corresponding to position F10 in thelambda light chain of SEQ ID NO:10 is not F, or

(ii) the amino acid in the position corresponding to position K55 in thelambda light chain of SEQ ID NO:10 is not K, or

(iii) the amino acid in the position corresponding to position F10 inthe lambda light chain of SEQ ID NO:10 is not F, and the amino acid inthe position corresponding to position K55 in the lambda light chain ofSEQ ID NO:10 is not K.

In one embodiment, the antibody according to the invention comprises aconstant light chain (LC), wherein

(i) the amino acid in the position corresponding to position F10 in thelambda light chain of SEQ ID NO:10 is L, or

(ii) the amino acid in the position corresponding to position K55 in thelambda light chain of SEQ ID NO:10 is N, or

(iii) the amino acid in the position corresponding to position F10 inthe lambda light chain of SEQ ID NO:10 is L, and the amino acid in theposition corresponding to position K55 in the lambda light chain of SEQID NO:10 is N.

In one embodiment, the antibody according to the invention comprises alight chain (LC), wherein the amino acids in the positions correspondingto positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO:10 are not F, T, K and L, respectively. Such modifications serve bothto increase the expression level and to reduce the affinity.

The antibody according to the invention comprises a light chain (LC),wherein the amino acids in the positions corresponding to positions F10,T41, K55, and L97 in the lambda light chain of SEQ ID NO:10 are L, K, N,and H, respectively. Such modifications serve both to increase theexpression level and to reduce the affinity.

In one embodiment, the antibody according to the invention comprises aheavy chain (HC) and a light chain (LC), wherein the positionscorresponding to positions L234, L235, and D265 in the human IgG1 heavychain of SEQ ID NO:15 are F, E, and A, respectively, and wherein theposition corresponding to F405 in in the human IgG1 heavy chain of SEQID NO:15 is L, and wherein (i) the positions corresponding to positionsF10, T41, K55, and L97 in the lambda light chain of SEQ ID NO:10 are L,K, N, and H, respectively, or (ii) the position corresponding toposition T41 in the lambda light chain of SEQ ID NO:10 is K.

In one embodiment, the antibody according to the invention comprises theheavy chain (HC) of SEQ ID NO:25 and the light chain (LC) of SEQ IDNO:32.

In one embodiment, the antibody according to the invention comprises theheavy chain (HC) of SEQ ID NO:25 and the light chain (LC) of SEQ IDNO:33.

In one aspect, the present invention relates to a multispecific antibodycomprising at least a first binding region of an antibody according toany aspect or embodiment herein described, and one or more bindingregions which binds one or more different targets than the first bindingregion. Such a multispecific antibody may be a bispecific antibody.

Thus, in one aspect, the present invention relates to a bispecificantibody comprising a first binding region of an antibody according toany aspect or embodiment herein described, and a second binding regionwhich binds a different target than the first binding region.

The term “multispecific antibody” refers to an antibody havingspecificities for at least two different, such as at least three,typically non-overlapping, epitopes. Such epitopes may be on the same ordifferent targets. If the epitopes are on different targets, suchtargets may be on the same cell or different cells or cell types.

The term “bispecific antibody” refers to an antibody havingspecificities for at least two different, typically non-overlapping,epitopes. Such epitopes may be on the same or different targets. If theepitopes are on different targets, such targets may be on the same cellor different cells or cell types.

In one embodiment, the bispecific antibody comprises a first and asecond heavy chain.

The embodiments relating to modification of the Fc region andembodiments relating to specific amino acid substitutions arecontemplated to be part of any bispecific antibody according to theinvention. Thus, in one embodiment, at least one of the first and secondheavy chains comprise one or more amino acids modified as defined in anyembodiment herein described, such as those described to in relation toproviding an inert Fc region. In one embodiment, both said first andsecond heavy chains comprise one or more amino acids modified as definedin any embodiment herein described, such as those described to inrelation to providing an inert Fc region. Accordingly, the bispecificantibody comprises an Fc region modified according to any aspect orembodiment herein described; or at least one of said first and secondheavy chains comprise one or more amino acids modified as defined in anyaspect or embodiment herein described.

Thus, in one embodiment, the bispecific antibody comprises a firstbinding region comprising a VH sequence as set out in SEQ ID NO:8 and aVL sequence as set out in SEQ ID NO:10; and wherein the Fc region hasbeen modified so that binding of C1q to said antibody is reducedcompared to a wild-type antibody by at least 70%, at least 80%, at least90%, at least 95%, at least 97%, or 100%, wherein C1q binding isdetermined by ELISA.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that binding of C1q to said antibody is reduced compared toa wild-type antibody by at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100%, wherein C1q binding is determined byELISA.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that binding of C1q to said antibody is reduced compared toa wild-type antibody by at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100%, wherein C1q binding is determined byELISA.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that binding of C1q to said antibody is reduced compared toa wild-type antibody by at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100%, wherein C1q binding is determined byELISA.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that binding of C1q to said antibody is reduced compared toa wild-type antibody by at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100%, wherein C1q binding is determined byELISA.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that binding of C1q to said antibody is reduced compared toa wild-type antibody by at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, or 100%, wherein C1q binding is determined byELISA.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody mediates reduced Fc-mediated T-cellproliferation compared to a wild-type antibody by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%,wherein said T-cell proliferation is measured in a peripheral bloodmononuclear cell (PBMC)-based functional assay.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:10; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:12; and wherein the Fc region has beenmodified so that said antibody reduces Fc-mediated CD69 expression by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100% when compared to a wild-type antibody wherein saidFc-mediated CD69 expression is determined in a PBMC-based functionalassay.

In a particular embodiment, the bispecific antibody comprises a firstbinding region comprising a VH sequence as set out in SEQ ID NO:8 and aVL sequence as set out in SEQ ID NO:10; and wherein in at least one ofthe first and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8 and a VLsequence as set out in SEQ ID NO:12; and wherein in at least one of thefirst and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:10; and wherein in at least one of thefirst and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6 and a VLsequence as set out in SEQ ID NO:12; and wherein in at least one of thefirst and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:10; and wherein in at least one of thefirst and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9 and a VLsequence as set out in SEQ ID NO:12; and wherein in at least one of thefirst and second heavy chains the amino acids in the positionscorresponding to positions L234, L235, and D265 in a human IgG1 heavychain, are F, E, and A, respectively.

Examples of bispecific antibody molecules which may be used in thepresent invention comprise (i) a single antibody that has two armscomprising different antigen-binding regions, (ii) a single chainantibody that has specificity to two different epitopes, e.g., via twoscFvs linked in tandem by an extra peptide linker; (iii) adual-variable-domain antibody (DVD-Ig™), where each light chain andheavy chain contains two variable domains in tandem through a shortpeptide linkage ([40]); (iv) a chemically-linked bispecific (Fab′)2fragment; (v) a TandAb®, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (vi) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(vii) a so called “dock and lock” molecule (Dock-and-Lock®), based onthe “dimerization and docking domain” in Protein Kinase A, which, whenapplied to Fabs, can yield a trivalent bispecific binding proteinconsisting of two identical Fab fragments linked to a different Fabfragment; (viii) a so-called Scorpion molecule, comprising, e.g., twoscFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

In one embodiment, the bispecific antibody of the present invention is adiabody, a cross-body, or a bispecific antibody obtained via acontrolled Fab arm exchange, e.g. DuoBody® (such as described in [41])as those described in the present invention.

Examples of different classes of bispecific antibodies include but arenot limited to (i) IgG-like molecules with complementary CH3 domains toforce heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of the molecule each contain the Fabfragment or part of the Fab fragment of at least two differentantibodies; (iii) IgG fusion molecules, wherein full length IgGantibodies are fused to extra Fab fragment or parts of Fab fragment;(iv) Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; (v) Fab fusion molecules, wherein differentFab-fragments are fused together, fused to heavy-chain constant-domains,Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, Nanobodies®) whereindifferent single chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, Nanobodies®) are fusedto each other or to another protein or carrier molecule fused toheavy-chain constant-domains, Fc-regions or parts thereof.

Examples of IgG-like molecules with complementary CH3 domains moleculesinclude but are not limited to the Triomab® (Trion Pharma/FreseniusBiotech, [42]), the Knobs-into-Holes (Genentech, [43]), CrossMAbs(Roche, [44]) and the electrostatically-matched (Amgen, [45]-[46];Chugai, [47]; Oncomed, [48]), the LUZ-Y (Genentech), DIG-body andPIG-body (Pharmabcine), the Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono, [49]), the Biclonics (Merus), FcΔAdp (Regeneron,[50]), bispecific IgG1 and IgG2 (Pfizer/Rinat, [51]), Azymetric scaffold(Zymeworks/Merck, [52]), mAb-Fv (Xencor, [53]), bivalent bispecificantibodies (Roche) and DuoBody® molecules (Genmab A/S, [41]).

Examples of recombinant IgG-like dual targeting molecules include butare not limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-oneAntibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2(F-Star, [54]), Zybodies™ (Zyngenia), approaches with common light chain(Crucell/Merus, [55]), KABodies (NovImmune) and CovX-body®(CovX/Pfizer).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig™ (Abbott, [56]), Dual domain double headantibodies (Unilever; Sanofi Aventis, [57]), IgG-like Bispecific(ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics),HERCULES (Biogen Idec, [58]), scFv fusion (Novartis), scFv fusion(Changzhou Adam Biotech Inc, [59]) and TvAb (Roche, [59], [60]).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART™)(MacroGenics, [62], [63]) and Dual(ScFv)2-Fab (National Research Centerfor Antibody Medicine—China).

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech).

Examples of ScFv-, diabody-based and domain antibodies include but arenot limited to Bispecific T Cell Engager (BITE®) (Micromet, TandemDiabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) andCOMBODY (Epigen Biotech), dual targeting Nanobodies® (Ablynx), dualtargeting heavy chain only domain antibodies.

It is further contemplated that any monospecific antibody fulfilling theassay conditions herein described may form the basis of a bispecificantibody. I.e. a bispecific antibody wherein one of the binding regionsbinds CD3 may originate from any monospecific CD3 antibody tested in thefunctional assays and fulfilling the requirements stated herein. Such abispecific antibody may be provided by the methods described in [41],which is hereby incorporated by reference.

Thus, in a particular embodiment, each of said first and second heavychain comprises at least a hinge region, a CH2 and CH3 region, whereinin said first heavy chain at least one of the amino acids in thepositions corresponding to a position selected from the group consistingof T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavychain has been substituted, and in said second heavy chain at least oneof the amino acids in the positions corresponding to a position selectedfrom the group consisting of T366, L368, K370, D399, F405, Y407, andK409 in a human IgG1 heavy chain has been substituted, and wherein saidfirst and said second heavy chains are not substituted in the samepositions. In this context the term “substituted”, refers to that theamino acid in a specific amino acid position has been substituted withanother naturally or non-naturally occurring amino acid. Thus, a“substituted” amino acid in a position corresponding to the position ina human IgG1 heavy chain means the amino acid at the particular positionis different from the naturally occurring amino acid in an IgG1 heavychain.

In one embodiment, in said first heavy chain the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M, and optionally the amino acid in the position corresponding toF405 in a human IgG1 heavy chain is F, and in said second heavy chain atleast one of the amino acids in the positions corresponding to aposition selected from the group consisting of; T366, L368, K370, D399,F405, and Y407 in a human IgG1 heavy chain has been substituted.

In one embodiment, in said first heavy chain the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M, and in said second heavy chain the amino acid in the positioncorresponding to F405 in a human IgG1 heavy chain is not F andoptionally the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is K.

In one embodiment, in said first heavy chain, the amino acid in theposition corresponding to F405 in a human IgG1 heavy chain is not F, R,and G, and in said second heavy chain the amino acids in the positionscorresponding to a position selected form the group consisting of; T366,L368, K370, D399, Y407, and K409 in a human IgG1 heavy chain has beensubstituted.

In one embodiment, the amino acid in position corresponding to K409 in ahuman IgG1 heavy chain is not K, L or M in said first heavy chain, andthe amino acid in position corresponding to F405 in a human IgG1 heavychain is not F.

In a further embodiment, the amino acid in the position corresponding toF405 in a human IgG1 heavy chain is L in said first heavy chain, and theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in said second heavy chain, or vice versa.

Thus, in one embodiment, the amino acid in the position corresponding toK409 in a human IgG1 heavy chain is R in the first heavy chain, and theamino acid in the position corresponding to F405 in a human IgG1 heavychain is L in the second heavy chain.

In a further embodiment, the humanized or chimeric CD3 antibody of theinvention contains in at least one of the first and second heavy chainone or more of the inactivating substitutions as disclosed in any one ofthe above embodiments, such as L234F, L235E, and D265A; and that theamino acid in the position corresponding to F405 is not F. In oneembodiment the humanized or chimeric CD3 antibody of the inventioncontains in at least one of the first and second heavy chain one or moreof the inactivating substitutions as disclosed in any one of the aboveembodiments, such as L234F, L235E, and D265A; and a further substitutionin the K409 position, such as K409R. In particular, in one embodiment,the humanized or chimeric CD3 antibody of the invention contains in boththe first and second heavy chain one or more of the inactivatingsubstitutions as disclosed in any one of the above embodiments, such asL234F, L235E, and D265A; and a substitution in the F405 position, suchas F405L. In one embodiment the humanized or chimeric CD3 antibody ofthe invention contains in both the first and second heavy chain one ormore of the inactivating substitutions as disclosed in any one of theabove embodiments, such as L234F, L235E, and D265A; and a furthersubstitution in the K409 position, such as K409R. Such antibodies areuseful for generating a bispecific antibody.

Accordingly, in a further embodiment, in at least one of the first andsecond heavy chains the amino acids in the positions corresponding toposition L234, L235, and D265 in a human IgG1 heavy chain are F, E, andA, respectively, the amino acid in the position corresponding to F405 ina human IgG1 heavy chain is L in the first heavy chain, and the aminoacid in the position corresponding to K409 in a human IgG1 heavy chainis R in the second heavy chain.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234, L235, D265,N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and Prespectively, the amino acid in the position corresponding to F405 in ahuman IgG1 heavy chain is L in the first heavy chain, and the amino acidin the position corresponding to K409 in a human IgG1 heavy chain is Rin the second heavy chain.

In an alternative embodiment, in at least one of the first and secondheavy chains the amino acids in the positions corresponding to positionL234, L235, and D265 in a human IgG1 heavy chain are F, E, and A,respectively, the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is R in the first heavy chain, and the amino acidin the position corresponding to F405 in a human IgG1 heavy chain is Lin the second heavy chain.

In one embodiment, in at least one of the first and second heavy chainsthe amino acids in the positions corresponding to L234, L235, D265,N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and Prespectively, the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is R in the first heavy chain, and the amino acidin the position corresponding to F405 in a human IgG1 heavy chain is Lin the second heavy chain.

In another embodiment, in both the first and second heavy chains theamino acids in the positions corresponding to position L234, L235, andD265 in a human IgG1 heavy chain are F, E, and A, respectively, theamino acid in the position corresponding to F405 in a human IgG1 heavychain is L in the first heavy chain, and the amino acid in the positioncorresponding to K409 in a human IgG1 heavy chain is R in the secondheavy chain.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234, L235, D265, N297, and P331in a human IgG1 heavy chain are F, E, A, N, and P respectively, theamino acid in the position corresponding to F405 in a human IgG1 heavychain is L in the first heavy chain, and the amino acid in the positioncorresponding to K409 in a human IgG1 heavy chain is R in the secondheavy chain.

In an alternative embodiment, in both the first and second heavy chainsthe amino acids in the positions corresponding to position L234, L235,and D265 in a human IgG1 heavy chain are F, E, and A, respectively, theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in the first heavy chain, and the amino acid in the positioncorresponding to F405 in a human IgG1 heavy chain is L in the secondheavy chain.

In one embodiment, in both the first and second heavy chains the aminoacids in the positions corresponding to L234, L235, D265, N297, and P331in a human IgG1 heavy chain are F, E, A, N, and P respectively, theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in the first heavy chain, and the amino acid in the positioncorresponding to F405 in a human IgG1 heavy chain is L in the secondheavy chain.

As described herein, T-cell recruitment to specific target cells, suchas cancer or tumor cells, provides a way of killing the target cells.The present inventors have shown that a bispecific CD3×HER2 antibodycomprising the specific amino acid substitutions L234F, L235E, and D265Ain both of the heavy chains, was able to induce killing of AU565 cellsas described in Example 5. T-cell mediated killing may be obtained by abispecific antibody targeting CD3 with the first binding region andanother target with the second binding region. Thus, in one embodiment,the first binding region is according to any embodiments describedherein for the humanized or chimeric CD3 antibody, and the secondbinding region binds a different target than the first binding region.It is to be understood that when the antibody is a bispecific antibody,at least one half of the antibody, i.e. one of the pair of heavy andlight chains of the antibody, is a humanized or chimeric antibody asherein described. Thus, one half of the bispecific antibody is ahumanized or chimeric antibody binding CD3 according to the presentinvention and the other half may be humanized, chimeric, fully non-humanor fully human binding a second target. Thus, in one embodiment, theantibody comprises a first and a second heavy chain, a first and secondlight chain, wherein said first heavy and said first light chains arehumanized or chimeric and are connected via disulfide bridges forming afirst binding region; and said second heavy and light chains are fullyhuman and are connected via disulfide bridges forming a second bindingregion, wherein said first binding region is according to any aspect orembodiment herein described, and said second binding region binds adifferent target. In one embodiment, the antibody comprises a first anda second heavy chain, a first and second light chain, wherein said firstheavy and said first light chains are humanized or chimeric and areconnected via disulfide bridges forming a first binding region; and saidsecond heavy and light chains are humanized or chimeric and areconnected via disulfide bridges forming a second binding region, whereinsaid first binding region is according to any aspect or embodimentherein described, and said second binding region binds a differentepitope of CD3 than said first binding region.

Thus, in one embodiment, the bispecific antibody comprises a firstbinding region comprising a VH sequence as set out in SEQ ID NO:8, and aVL sequence as set out in SEQ ID NO:10; a second binding regioncomprising a VH sequence as set out in SEQ ID NO:19, and a VL sequenceas set out in SEQ ID NO:20; wherein in at least one of the first andsecond heavy chains the amino acids in the positions corresponding topositions L234, L235, and D265 in a human IgG1 heavy chain, are F, E,and A, respectively; and wherein in the first heavy chain the amino acidin the position corresponding to position F405 in a human IgG1 heavychain, is L, and in the second heavy chain the amino acid in theposition corresponding to position K409 in a human IgG1 heavy chain, isR.

In one embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:8, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:6, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:10; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in at least one of the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively; and wherein in the first heavy chain the amino acid in theposition corresponding to position F405 in a human IgG1 heavy chain, isL, and in the second heavy chain the amino acid in the positioncorresponding to position K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:19, and a VL sequence as set outin SEQ ID NO:20; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

In another embodiment, the bispecific antibody comprises a first bindingregion comprising a VH sequence as set out in SEQ ID NO:9, and a VLsequence as set out in SEQ ID NO:12; a second binding region comprisinga VH sequence as set out in SEQ ID NO:29, and a VL sequence as set outin SEQ ID NO:30; wherein in both the first and second heavy chains theamino acids in the positions corresponding to positions L234, L235, andD265 in a human IgG1 heavy chain, are F, E, and A, respectively; andwherein in the first heavy chain the amino acid in the positioncorresponding to position F405 in a human IgG1 heavy chain, is L, and inthe second heavy chain the amino acid in the position corresponding toposition K409 in a human IgG1 heavy chain, is R.

The term “disulfide bridges” as used herein refers to the covalent bondbetween two Cysteine residues, i.e. said interaction may also bedesignated a Cys-Cys interaction.

The term “target” as used herein, refers to a molecule to which thebinding region of the antibody according to the invention binds. Whenused in the context of the binding of an antibody the term includes anyantigen towards which the raised antibody is directed.

In one particular embodiment, the first heavy and the first light chainsare humanized or chimeric and are connected via disulfide bridgesforming a first binding region; and the second heavy and light chainsare fully human and are connected via disulfide bridges forming a secondbinding region, wherein the first binding region is according to anyaspect or embodiment herein described, and the second binding regionbinds a different target; and wherein in at least one of the first andsecond heavy chains the amino acids in the positions corresponding topositions L234, L235, and D265 in a human IgG1 heavy chain, are F, E,and A, respectively.

In one particular embodiment, the first heavy and the first light chainsare humanized or chimeric and are connected via disulfide bridgesforming a first binding region; and the second heavy and light chainsare fully human and are connected via disulfide bridges forming a secondbinding region, wherein the first binding region is according to anyaspect or embodiment herein described, and the second binding regionbinds a different epitope of CD3 than the first binding region; andwherein in at least one of the first and second heavy chains the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain, are F, E, and A, respectively.

In one particular embodiment, the first heavy and the first light chainsare humanized or chimeric and are connected via disulfide bridgesforming a first binding region; and the second heavy and light chainsare fully human and are connected via disulfide bridges forming a secondbinding region, wherein the first binding region is according to anyaspect or embodiment herein described, and the second binding regionbinds a different target; and wherein in both the first and second heavychains the amino acids in the positions corresponding to positions L234,L235, and D265 in a human IgG1 heavy chain, are F, E, and A,respectively.

In one particular embodiment, the first heavy and the first light chainsare humanized or chimeric and are connected via disulfide bridgesforming a first binding region; and the second heavy and light chainsare fully human and are connected via disulfide bridges forming a secondbinding region, wherein the first binding region is according to anyaspect or embodiment herein described, and the second binding regionbinds a different epitope of CD3 than the first binding region; andwherein in both the first and second heavy chains the amino acids in thepositions corresponding to positions L234, L235, and D265 in a humanIgG1 heavy chain, are F, E, and A, respectively.

In one aspect, the bispecific antibody according to the inventioncomprises a second binding region which binds to human HER2 or humanCD20.

In one embodiment, the bispecific antibody according to the inventioncomprises a second binding region which binds to human CD20.

In one embodiment, the second binding region may be derived from afull-length monoclonal CD20 antibody, such as an antibody disclosed inWO2004035607 (Genmab A/S), including ofatumumab (2F2), 11B8, and7D8; anantibody disclosed in WO2005103081 (Genmab A/S), including 2C6; AME-133;TRU-015; IMMU-106; ocrelizumab (Gazyva®); tositumomab (Bexxar®); andrituximab (Rituxan®/MabThera®).

In one embodiment, the second binding region may be derived from afull-length monoclonal CD20 antibody, which antibody binds to an epitopeon CD20, which does not comprise or require the amino acid residuesalanine at position 170 or proline at position 172, but which comprisesor requires the amino acid residues asparagine at position 163 andasparagine at position 166.

In one embodiment, the bispecific antibody according to the inventioncomprises a second binding region binding to human CD20, which bindingregion comprises:

(i) the VH CDR1 region of SEQ ID NO:34, the VH CDR2 region of SEQ IDNO:35, the VH CDR3 region of SEQ ID NO:36, the VL CDR1 region of SEQ IDNO:37, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ IDNO:38,

(ii) the VH CDR1 region of SEQ ID NO: 41, the VH CDR2 region of SEQ IDNO:42, the VH CDR3 region of SEQ ID NO:43, the VL CDR1 region of SEQ IDNO:44, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ IDNO:45,

(iii) the VH CDR1 region of SEQ ID NO:48, the VH CDR2 region of SEQ IDNO:49, the VH CDR3 region of SEQ ID NO:50, the VL CDR1 region of SEQ IDNO:51, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ IDNO:52, or

(iv) the VH CDR1 region of SEQ ID NO:55, the VH CDR2 region of SEQ IDNO:56, the VH CDR3 region of SEQ ID NO:57, the VL CDR1 region of SEQ IDNO:58, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ IDNO:59.

In one embodiment, the bispecific antibody according to the inventioncomprises a second binding region binding to human CD20, which bindingregion comprises:

(i) a VH sequence which has at least 90%, at least 95%, at least 97%, orat least 99% amino acid sequence identity to the amino acid sequence asset forth in SEQ ID NO:29, and a VL sequence which has at least 90%, atleast 95%, at least 97%, or at least 99% amino acid sequence identity tothe amino acid sequence as set forth in SEQ ID NO:30,

(ii) a VH sequence which has at least 90%, at least 95%, at least 97%,or at least 99% amino acid sequence identity to the amino acid sequenceas set forth in SEQ ID NO:39, and a VL sequence which has at least 90%,at least 95%, at least 97%, or at least 99% amino acid sequence identityto the amino acid sequence as set forth in SEQ ID NO:40,

(iii) a VH sequence which has at least 90%, at least 95%, at least 97%,or at least 99% amino acid sequence identity to the amino acid sequenceas set forth in SEQ ID NO:46, and a VL sequence which has at least 90%,at least 95%, at least 97%, or at least 99% amino acid sequence identityto the amino acid sequence as set forth in SEQ ID NO:47, or

(iv) a VH sequence which has at least 90%, at least 95%, at least 97%,or at least 99% amino acid sequence identity to the amino acid sequenceas set forth in SEQ ID NO:53, and a VL sequence which has at least 90%,at least 95%, at least 97%, or at least 99% amino acid sequence identityto the amino acid sequence as set forth in SEQ ID NO:54.

In one embodiment, the bispecific antibody according to the inventioncomprises a second binding region binding to human CD20, which secondbinding region comprises:

(i) the VH sequence of SEQ ID NO: 29 and the VL sequence of SEQ IDNO:30,

(ii) the VH sequence of SEQ ID NO:39, and the VL sequence of SEQ IDNO:40,

(iii) the VH sequence of SEQ ID NO:46, and the VL sequence of SEQ IDNO:47, or

(iv) the VH sequence of SEQ ID NO:53, and the VL sequence of SEQ IDNO:54.

Nucleic Acid Constructs, Expression Vectors, and Host Cells

In one aspect, the present invention relates to a nucleic acid constructencoding one or more sequences set out in Table 1. Thus, the presentinvention relates to a nucleic acid construct encoding any one of thesequences set out in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.

In a further aspect, the invention relates to nucleic acid constructencoding a sequence of a humanized or chimeric CD3 antibody according tothe present invention, to expression vectors comprising a nucleic acidconstruct according to the present invention, to host cells comprisingsuch expression vectors, and to methods of producing such an antibody byculturing such host cells under appropriate conditions whereby theantibody is produced and, optionally, retrieved. Humanized CD3antibodies may also be denoted as “huCD3”.

In one embodiment, the invention provides an expression vectorcomprising (i) a nucleic acid sequence encoding a heavy chain sequenceof a humanized or chimeric antibody according to the invention, (ii) anucleic acid sequence encoding a light chain sequence of a humanized orchimeric antibody according to the invention, or (iii) both (i) and(ii). Thus, the expression vector comprises one or more nucleic acidconstructs or nucleic acid sequences according to any aspect orembodiment herein described.

In one embodiment, the expression vector of the invention comprises anucleic acid sequence encoding one or more of the heavy chain and lightchain CDR sequences selected from the group consisting of: SEQ IDNOs.:1, 2, 3, 4, and 5; and the sequence GTN.

In one embodiment, the invention provides an expression vectorcomprising a nucleic acid sequence encoding one or more amino acidsequences selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9,10, 11, 12, 19, 20, 27, 28, 29, and 30, or any combination thereof. Inanother embodiment, the expression vector comprises a nucleic acidsequence encoding the VH CDR3 amino acid sequence as set forth in SEQ IDNO: 3. In another embodiment, the expression vector comprises a nucleicacid sequence encoding a VH amino acid sequence selected from SEQ IDNOs: 6, 7, 8, 9, 19, 27, and 29. In another embodiment, the expressionvector comprises a nucleic acid sequence encoding a VL amino acidsequence selected from SEQ ID NOs: 10, 11, 12, 20, 28, and 30. Inanother embodiment, the expression vector comprises a nucleic acidsequence encoding the constant region of a human antibody light chain,of a human antibody heavy chain, or both. In another embodiment, theinvention provides an expression vector comprising a nucleic acidsequence encoding the amino acid sequence according to SEQ ID NOs: 15,16, 23, 24, 25, and 26.

In a particular embodiment, the expression vector comprises a nucleicacid sequence encoding a variant of one or more of the above amino acidsequences, said variant having at most 25 amino acid modifications, suchas at most 20, such as at most 15, 14, 13, 12, or 11 amino acidmodifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidmodifications, such as deletions or insertions, preferablysubstitutions, such as conservative or non-conservative substitutions,or at least 80% identity to any of said sequences, such as at least 85%identity or 90% identity or 95% identity, such as 96% identity or 97%identity or 98% identity or 99% identity to any of the afore-mentionedamino acid sequences. The present invention also relates to nucleic acidsequences different from the above mentioned nucleic acid sequences butwhich due to the variance of the genetic code encode the same amino acidsequence as an antibody of the present invention. E.g. the nucleic acidsequence may vary but result in an identical amino acid sequences as anyamino acid sequence herein described. It is well-known for the skilledperson how to identify such further nucleic acid sequences based on thegenetic code.

In a further embodiment, the expression vector further comprises anucleic acid sequence encoding the constant region of a light chain, aheavy chain or both light and heavy chains of an antibody, e.g. a humanantibody.

Such expression vectors as described above may be used for recombinantproduction of antibodies of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ahumanized or chimeric CD3 antibody-encoding nucleic acid is comprised ina naked DNA or RNA vector, including, for example, a linear expressionelement (as described in for instance [64]), a compacted nucleic acidvector (as described in for instance [65] and/or [66]), a plasmid vectorsuch as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sizednucleic acid vector (as described in for instance [67]), or as aprecipitated nucleic acid vector construct, such as a CaPO₄⁻-precipitated construct (as described in for instance [68], [69], [70],and [71]). Such nucleic acid vectors and the usage thereof are wellknown in the art (see for instance [72] and [73]).

In one embodiment, the vector is suitable for expression of thehumanized or chimeric CD3 antibody in a bacterial cell. Examples of suchvectors include expression vectors such as BlueScript (Stratagene), pINvectors ([74]), pET vectors (Novagen, Madison Wis.) and the like.

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: [75] and [76]).

A nucleic acid construct and/or vector may also comprise a nucleic acidsequence encoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the art,and include secretion leader or signal peptides, organelle-targetingsequences (e. g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e. g., stop transfer sequences,GPI anchor sequences), and the like which are well-known in the art.

In an expression vector of the invention, humanized or chimeric CD3antibody-encoding nucleic acids may comprise or be associated with anysuitable promoter, enhancer, and other expression-facilitating elements.Examples of such elements include strong expression promoters (e. g.,human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, andHIV LTR promoters), effective poly (A) termination sequences, an originof replication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acid constructs and/or vectors may also comprise aninducible promoter as opposed to a constitutive promoter such as CMV IE(the skilled person will recognize that such terms are actuallydescriptors of a degree of gene expression under certain conditions).

In one embodiment, the humanized or chimeric CD3 antibody-encodingexpression vector is positioned in and/or delivered to the host cell orhost animal via a viral vector.

Such expression vectors may be used for recombinant production ofhumanized or chimeric CD3 antibodies.

In one aspect, the invention provides a host cell comprising anexpression vector according to the invention.

In one aspect, the humanized or chimeric CD3 antibodies of any aspect orembodiment described herein are provided by use of recombinanteukaryotic, recombinant prokaryotic, or recombinant microbial host cellwhich produces the antibody. Accordingly, the invention provides arecombinant eukaryotic, recombinant prokaryotic, or recombinantmicrobial host cell, which produces a humanized or chimeric CD3 antibodyor immunoglobulin as defined herein. Examples of host cells includeyeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. Forexample, in one embodiment, the host cell comprises a nucleic acidsequence stably integrated into the cellular genome that comprises asequence coding for expression of a humanized or chimeric CD3 antibodydescribed herein. In another embodiment, the host cell comprises anon-integrated nucleic acid sequence, such as a plasmid, cosmid,phagemid, or linear expression element, which comprises a sequencecoding for expression of a humanized or chimeric CD3 antibody describedherein.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectoror nucleic acid construct or sequence has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell, but also to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Recombinant host cells include, forexample, eukaryotic host cells, such as CHO cells, HEK-293 cells,PER.C6, NS0 cells, and lymphocytic cells, and prokaryotic cells such asE. coli and other eukaryotic hosts such as plant cells and fungi.

In a further aspect, the invention relates to a method for producing ahumanized or chimeric CD3 antibody of the invention, said methodcomprising the steps of

a) culturing a host cell of the invention as described herein above, and

b) retrieving and/or purifying the antibody of the invention from theculture media.

In a further aspect, the nucleotide sequence encoding a sequence of ahumanized or chimeric CD3 antibody further encodes a second moiety, suchas a therapeutic polypeptide. Exemplary therapeutic polypeptides aredescribed elsewhere herein. In one embodiment, the invention relates toa method for producing a humanized or chimeric CD3 antibody fusionprotein, said method comprising the steps of

a) culturing a host cell comprising an expression vector comprising sucha nucleotide sequence, and

b) retrieving and/or purifying the humanized or chimeric CD3 antibodyfusion protein from the culture media.

Compositions

In one aspect, the invention provides a composition comprising theantibody or bispecific antibody according to any aspect and embodimentherein described.

In one aspect, the invention provides a pharmaceutical compositioncomprising the antibody or bispecific antibody as defined in any one ofthe aspects and embodiments herein described, and a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in [77].

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for thehumanized or chimeric antibody of the present invention and the chosenmode of administration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition of the present invention(e.g., less than a substantial impact (10% or less relative inhibition,5% or less relative inhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e. g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the amide thereof, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a humanized or chimericantibody of the present invention in vivo and in vitro are well known inthe art and may be selected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intra-arterial, intrathecal, intracapsular,intra-orbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption-delaying agents,and the like that are physiologically compatible with a humanized orchimeric antibody of the present invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated. When referring to the “activecompound” it is contemplated to also refer to the humanized or chimericantibody according to the present invention.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thehumanized or chimeric antibody of the present invention may be preparedwith carriers that will protect the compound against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and micro-encapsulated delivery systems. Such carriers mayinclude gelatin, glyceryl monostearate, glyceryl distearate,biodegradable, biocompatible polymers such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, poly-orthoesters, andpolylactic acid alone or with a wax, or other materials well known inthe art. Methods for the preparation of such formulations are generallyknown to those skilled in the art (see e.g., [78]).

In one embodiment, the humanized or chimeric antibody of the presentinvention may be formulated to ensure proper distribution in vivo.Pharmaceutically acceptable carriers for parenteral administrationinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Other active or therapeutic compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or a non-aqueous solvent or dispersion medium containingfor instance water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum-drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Therapeutic Applications

In another aspect, the present invention relates to a humanized orchimeric antibody, or pharmaceutical composition of the invention asdefined in any aspect or embodiment herein described, for use as amedicament.

In another aspect, the present invention relates to a humanized orchimeric antibody, or pharmaceutical composition of the invention asdefined in any aspect or embodiment herein described, for use in thetreatment of a disease.

The humanized or chimeric antibody or pharmaceutical composition of theinvention can be used as in the treatment of any cancer wherein theeffector mechanisms of cytotoxic T-cells are desired. For example, thehumanized or chimeric antibody may be administered to cells in culture,e.g., in vitro or ex vivo, or to human subjects, e.g. in vivo, to treator prevent disorders such as cancer, inflammatory or autoimmunedisorders. As used herein, the term “subject” is typically a human whichrespond to the humanized or chimeric antibody, or pharmaceuticalcomposition. Subjects may for instance include human patients havingdisorders that may be corrected or ameliorated by modulating a targetfunction or by leading to killing of the cell, directly or indirectly.

In another aspect, the present invention provides methods for treatingor preventing a disorder, such as cancer, wherein recruitment of T-cellswould contribute to the treatment or prevention, which method comprisesadministration of a therapeutically effective amount of a humanized orchimeric antibody, or pharmaceutical composition of the presentinvention to a subject in need thereof. The method typically involvesadministering to a subject a humanized or chimeric antibody in an amounteffective to treat or prevent the disorder.

In one particular aspect, the present invention relates to a method oftreatment of cancer comprising administering the humanized or chimericantibody or pharmaceutical composition of the invention as defined inany aspect and embodiments herein described, to a subject in needthereof.

In another aspect, the present invention relates to the use or themethod as defined in any aspect or embodiments herein described whereinthe humanized or chimeric antibody is a bispecific antibody specificallybinding to both CD3 and a cancer-specific target, or a target that isoverexpressed in cancer or associated with cancer, such as HER2, CD19,EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA)and wherein the disease is cancer, such as breast cancer, prostatecancer, non-small cell lung cancer, bladder cancer, ovarian cancer,gastric cancer, colorectal cancer, esophageal cancer and squamous cellcarcinoma of the head & neck, cervical cancer, pancreatic cancer, testiscancer, malignant melanoma, a soft-tissue cancer (e.g., synovialsarcoma), an indolent or aggressive form of B-cell lymphoma, chroniclymphatic leukemia or acute lymphatic leukemia.

The efficient dosages and dosage regimens for the humanized or chimericantibody depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of the humanizedor chimeric antibody employed in the pharmaceutical composition atlevels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, a suitable dose of a composition of thepresent invention will be that amount of the humanized or chimericantibody which is the lowest dose effective to produce a therapeuticeffect according to a particular dosage regimen. Such an effective dosewill generally depend upon the factors described above.

For example, an “effective amount” for therapeutic use may be measuredby its ability to stabilize the progression of disease. The ability of acompound to inhibit cancer may, for example, be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition may be evaluated by examining the ability ofthe humanized or chimeric antibody to inhibit cell growth or to inducecytotoxicity by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound, i.e. atherapeutic humanized or chimeric antibody, or pharmaceuticalcomposition according to the invention, may decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

An exemplary, non-limiting range for a therapeutically effective amountof a humanized or chimeric antibody of the invention is about 0.001-30mg/kg, such as about 0.001-20 mg/kg, such as about 0.001-10 mg/kg, suchas about 0.001-5 mg/kg, for example about 0.001-2 mg/kg, such as about0.001-1 mg/kg, for instance about 0.001, about 0.01, about 0.1, about 1,about 5, about 8, about 10, about 12, about 15, about 18 mg/kg.

Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous, and for instance administered proximal to the site ofthe target.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

In one embodiment, the efficacy of the treatment is monitored during thetherapy, e.g. at predefined points in time.

If desired, an effective daily dose of a pharmaceutical composition maybe administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In another embodiment, the humanizedor chimeric antibody, or pharmaceutical composition is administered byslow continuous infusion over a long period, such as more than 24 hours,in order to minimize any unwanted side effects.

While it is possible for a humanized or chimeric antibody of the presentinvention to be administered alone, it is preferable to administer thehumanized or chimeric antibody as a pharmaceutical composition asdescribed above.

An effective dose of a humanized or chimeric antibody of the inventionmay also be administered using a weekly, biweekly or triweekly dosingperiod. The dosing period may be restricted to, e.g., 8 weeks, 12 weeksor until clinical progression has been established. Alternatively, aneffective dose of a humanized or chimeric antibody of the invention maybe administered every second, third or fourth week.

In one embodiment, the humanized or chimeric antibody may beadministered by infusion in a weekly dosage of calculated by mg/m². Suchdosages can, for example, be based on the mg/kg dosages provided aboveaccording to the following: dose (mg/kg)×70: 1.8. Such administrationmay be repeated, e.g., 1 to 8 times, such as 3 to 5 times. Theadministration may be performed by continuous infusion over a period offrom 2 to 24 hours, such as of from 2 to 12 hours. In one embodiment,the humanized or chimeric antibody may be administered by slowcontinuous infusion over a long period, such as more than 24 hours, inorder to reduce toxic side effects.

In one embodiment, the humanized or chimeric antibody may beadministered in a weekly dosage of calculated as a fixed dose for up to8 times, such as from 4 to 6 times when given once a week. Such regimenmay be repeated one or more times as necessary, for example, after 6months or 12 months. Such fixed dosages can, for example, be based onthe mg/kg dosages provided above, with a body weight estimate of 70 kg.The dosage may be determined or adjusted by measuring the amount ofhumanized or chimeric antibody of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the binding region of thehumanized or chimeric antibodies of the present invention.

In one embodiment, the humanized or chimeric antibody may beadministered by maintenance therapy, such as, e.g., once a week for aperiod of 6 months or more.

A humanized or chimeric antibody may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subjects to be treated; each unit contains a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

A humanized or chimeric antibody may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission. This may beespecially useful in patients wherein it is difficult to locate a tumorthat is known to be present due to other biological factors.

Diagnostic Applications

The humanized or chimeric antibody of the invention may also be used fordiagnostic purposes, using a composition comprising a humanized orchimeric antibody as described herein. Accordingly, the inventionprovides diagnostic methods and compositions using the humanized orchimeric antibodies described herein. Such methods and compositions canbe used for purely diagnostic purposes, such as detecting or identifyinga disease, as well as for monitoring of the progress of therapeutictreatments, monitoring disease progression, assessing status aftertreatment, monitoring for recurrence of disease, evaluating risk ofdeveloping a disease, and the like.

In one aspect, the present invention relates to a method of diagnosing adisease characterized by involvement or accumulation of CD3-expressioncells, comprising administering the humanized or chimeric antibodyaccording to the invention, the composition according to the invention,or the pharmaceutically composition according to the invention to asubject, optionally wherein said humanized or chimeric antibody islabeled with a detectable agent.

In one aspect, the humanized or chimeric antibody of the presentinvention is used ex vivo, such as in diagnosing a disease in whichcells expressing a specific target of interest and to which thehumanized or chimeric antibody binds, are indicative of disease orinvolved in the pathogenesis, by detecting levels of the target orlevels of cells which express the target of interest on their cellsurface in a sample taken from a patient. This may be achieved, forexample, by contacting the sample to be tested, optionally along with acontrol sample, with the humanized or chimeric antibody according to theinvention under conditions that allow for binding of the antibody to thetarget. Complex formation can then be detected (e.g., using an ELISA).When using a control sample along with the test sample, the level ofhumanized or chimeric antibody or antibody-target complex is analyzed inboth samples and a statistically significant higher level of humanizedor chimeric antibody or antibody-target complex in the test sampleindicates a higher level of the target in the test sample compared withthe control sample.

Examples of conventional immunoassays in which humanized or chimericantibodies of the present invention can be used include, withoutlimitation, ELISA, RIA, FACS assays, plasmon resonance assays,chromatographic assays, tissue immunohistochemistry, Western blot,and/or immunoprecipitation.

Accordingly, in one embodiment, the present invention relates to amethod of diagnosing a disease characterized by involvement oraccumulation of CD3-expressing cells, comprising administering anantibody, bispecific antibody, composition or pharmaceutical compositionaccording to any aspect or embodiment herein described, to a subject,optionally wherein the antibody is labeled with a detectable label.

In one embodiment, the invention relates to a method for detecting thepresence of a target, or a cell expressing the target, in a samplecomprising:

-   -   contacting the sample with a humanized or chimeric antibody of        the invention under conditions that allow for binding of the        humanized or chimeric antibody to the target in the sample; and    -   analyzing whether a complex has been formed. Typically, the        sample is a biological sample.

In one embodiment, the sample is a tissue sample known or suspected ofcontaining a specific target and/or cells expressing the target. Forexample, in situ detection of the target expression may be accomplishedby removing a histological specimen from a patient, and providing thehumanized or chimeric antibody of the present invention to such aspecimen. The humanized or chimeric antibody may be provided by applyingor by overlaying the humanized or chimeric antibody to the specimen,which is then detected using suitable means. It is then possible todetermine not only the presence of the target or target-expressingcells, but also the distribution of the target or target-expressingcells in the examined tissue (e.g., in the context of assessing thespread of cancer cells). Using the present invention, those of ordinaryskill will readily perceive that any of a wide variety of histologicalmethods (such as staining procedures) may be modified in order toachieve such in situ detection.

In the above assays, the humanized or chimeric antibody can be labeledwith a detectable substance to allow bound antibody to be detected.Alternatively, bound (primary) specific humanized or chimeric antibodymay be detected by an antibody which is labeled with a detectablesubstance and which binds to the primary specific humanized or chimericantibody. Furthermore, in the above assays, a diagnostic compositioncomprising an antibody or bispecific antibody according to any aspect orembodiments herein described may be used. Thus, in one aspect, thepresent invention relates to a diagnostic composition comprising anantibody or bispecific antibody according to any aspect or embodimentherein described.

The level of target in a sample can also be estimated by a competitionimmunoassay utilizing target standards labeled with a detectablesubstance and an unlabeled target-specific humanized or chimericantibody. In this type of assay, the biological sample, the labeledtarget standard(s) and the target-specific humanized or chimericantibody are combined, and the amount of labeled target standard boundto the unlabeled target-specific humanized or chimeric antibody isdetermined. The amount of target in the biological sample is inverselyproportional to the amount of labeled target standard bound to thetarget-specific humanized or chimeric antibody.

Suitable labels for the target-specific humanized or chimeric antibody,secondary antibody and/or target standard used in in vitro diagnostictechniques include, without limitation, various enzymes, prostheticgroups, fluorescent materials, luminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, and acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a luminescent materialincludes luminol; and examples of suitable radioactive material include¹²⁵I, ¹³¹I, ³⁵S, and ³H.

In one aspect, the target-specific humanized or chimeric antibody of theinvention is used in the in vivo imaging of target-expressing tissuessuch as tumors. For in vivo methods, antibody fragments such as, e.g.,(Fab′)₂, Fab and Fab′ fragments, are particularly advantageous becauseof their rapid distribution kinetics.

In vivo imaging can be performed by any suitable technique. For example,a target-specific humanized or chimeric antibody (e.g., an antibody or afragment) labeled with ⁹⁹Tc, ¹³¹I, ¹¹¹In or other gamma-ray emittingisotope may be used to image target-specific antibody accumulation ordistribution in target-expressing tissues such as tumors with a gammascintillation camera (e.g., an Elscint Apex 409ECT device), typicallyusing low-energy, high resolution collimator or a low-energy all-purposecollimator. Alternatively, labeling with ⁸⁹Zr, ⁷⁶Br, ¹⁸F or otherpositron-emitting radionuclide may be used to image target-specifichumanized or chimeric antibody, or antibody fragment distribution intumors using positron emission tomography (PET). The images obtained bythe use of such techniques may be used to assess biodistribution oftarget in a patient, mammal, or tissue, for example in the context ofusing target as a biomarker for the presence of cancer/tumor cells.Variations on this technique may include the use of magnetic resonanceimaging (MRI) to improve imaging over gamma camera techniques.Conventional immunoscintigraphy methods and principles are described in,e.g., [79], [80], and [81]. Moreover, such images may also, oralternatively, serve as the basis for surgical techniques to removetumors. Furthermore, such in vivo imaging techniques may allow for theidentification and localization of a tumor in a situation where apatient is identified as having a tumor (due to the presence of otherbiomarkers, metastases, etc.), but the tumor cannot be identified bytraditional analytical techniques. All of these methods are features ofthe present invention.

The in vivo imaging and other diagnostic methods provided by the presentinvention are particularly useful in the detection of micrometastases ina human patient (e.g., a patient not previously diagnosed with cancer ora patient in a period of recovery/remission from a cancer).

In one embodiment, the present invention provides an in vivo imagingmethod wherein a target-specific humanized or chimeric antibody of thepresent invention is conjugated to a detection-promoting radio-opaqueagent, the conjugated humanized or chimeric antibody is administered toa host, such as by injection into the bloodstream, and the presence andlocation of the labeled humanized or chimeric antibody in the host isassayed. Through this technique and any other diagnostic method providedherein, the present invention provides a method for screening for thepresence of disease-related cells in a human patient or a biologicalsample taken from a human patient and/or for assessing the distributionof target-specific humanized or chimeric antibody prior totarget-specific ADC therapy.

For diagnostic imaging, radioisotopes may be bound to a target-specifichumanized or chimeric antibody either directly or indirectly by using anintermediary functional group. Useful intermediary functional groupsinclude chelators, such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid (see for instance [82]).

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using target-specific antibodies that are conjugated todyes (such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g.,[83], which describes MRI techniques and the preparation of antibodiesconjugated to a MRI enhancing agent). Such diagnostic/detection agentsmay be selected from agents for use in MRI, and fluorescent compounds.In order to load a target-specific humanized or chimeric antibody withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which a multiplicity of chelatinggroups are attached for binding the ions. Such a tail may be a polymersuch as a polylysine, polysaccharide, or another derivatized orderivatizable chain having pendant groups to which may be boundchelating groups such as, e.g., porphyrins, polyamines, crown ethers,bisthiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates may be coupled to target-specific humanizedor chimeric antibodies using standard chemistries.

Thus, the present invention provides a diagnostic target-specifichumanized or chimeric antibody, wherein the target-specific humanized orchimeric antibody is conjugated to a contrast agent (such as formagnetic resonance imaging, computed tomography, or ultrasoundcontrast-enhancing agent) or a radionuclide that may be, for example, agamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope.

In one aspect, the present invention relates to a diagnostic compositioncomprising an antibody or bispecific antibody according to theinvention.

In a further aspect, the invention relates to a kit for detecting thepresence of target antigen or a cell expressing the target, in a sample,comprising:

-   -   a target-specific humanized or chimeric antibody of the        invention; and    -   instructions for use of the kit.

Thus, in one aspect, the present invention provides a kit for detectingthe presence of a CD3 antigen, or a cell expressing CD3, in a samplecomprising the steps of;

a) contacting the sample with an antibody or bispecific antibodyaccording to the invention, under conditions that allow for formation ofa complex between the antibody or bispecific antibody and CD3; andb) analyzing whether a complex has been formed.

In one embodiment, the present invention provides a kit for diagnosis ofcancer comprising a container comprising a target-specific humanized orchimeric antibody, and one or more reagents for detecting binding of thetarget-specific humanized or chimeric antibody to the target. Reagentsmay include, for example, fluorescent tags, enzymatic tags, or otherdetectable tags. The reagents may also include secondary or tertiaryantibodies or reagents for enzymatic reactions, wherein the enzymaticreactions produce a product that may be visualized. In one embodiment,the present invention provides a diagnostic kit comprising one or moretarget-specific humanized or chimeric antibodies of the presentinvention in labeled or unlabeled form in suitable container(s),reagents for the incubations for an indirect assay, and substrates orderivatizing agents for detection in such an assay, depending on thenature of the label. Control reagent(s) and instructions for use alsomay be included.

Diagnostic kits may also be supplied for use with a target-specifichumanized or chimeric antibody, such as a labeled target-specificantibody, for the detection of the presence of the target in a tissuesample or host. In such diagnostic kits, as well as in kits fortherapeutic uses described elsewhere herein, a target-specific humanizedor chimeric antibody typically may be provided in a lyophilized form ina container, either alone or in conjunction with additional antibodiesspecific for a target cell or peptide. Typically, a pharmaceuticallyacceptable carrier (e.g., an inert diluent) and/or components thereof,such as a Tris, phosphate, or carbonate buffer, stabilizers,preservatives, biocides, inert proteins, e.g., serum albumin, or thelike, also are included (typically in a separate container for mixing)and additional reagents (also typically in separate container(s)). Incertain kits, a secondary antibody capable of binding to thetarget-specific humanized or chimeric antibody, which typically ispresent in a separate container, is also included. The second antibodyis typically conjugated to a label and formulated in a manner similar tothe target-specific humanized or chimeric antibody of the presentinvention. Using the methods described above and elsewhere herein,target-specific humanized or chimeric antibodies may be used to definesubsets of cancer/tumor cells and characterize such cells and relatedtumor tissues.

Anti-Idiotypic Antibodies

In a further aspect, the invention relates to an anti-idiotypic antibodywhich binds to a humanized or chimeric antibody of the invention asdescribed herein.

An anti-idiotypic (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An anti-Id antibody may be prepared by immunizing an animal ofthe same species and genetic type as the source of a CD3 monoclonalantibody with the monoclonal antibody to which an anti-Id is beingprepared. The immunized animal typically can recognize and respond tothe idiotypic determinants of the immunizing antibody by producing anantibody to these idiotypic determinants (the anti-Id antibody). Suchantibodies are described in for instance U.S. Pat. No. 4,699,880. Suchantibodies are further features of the present invention.

An anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. An anti-anti-Id antibody may be epitopicallyidentical to the original monoclonal antibody, which induced the anti-Idantibody. Thus, by using antibodies to the idiotypic determinants of amonoclonal antibody, it is possible to identify other clones expressingantibodies of identical specificity. Anti-Id antibodies may be varied(thereby producing anti-Id antibody variants) and/or derivatized by anysuitable technique, such as those described elsewhere herein withrespect to CD3-specific antibodies of the present invention. Forexample, a monoclonal anti-Id antibody may be coupled to a carrier suchas keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice.Sera from these mice typically will contain anti-anti-Id antibodies thathave the binding properties similar, if not identical, to anoriginal/parent CD3 antibody.

Sequences

TABLE 1 SEQ ID NO: Clone name Sequence SEQ ID NO: 1 huCD3 VH CDR1GFTFNTYA SEQ ID NO: 2 huCD3 VH CDR2 IRSKYNNYAT SEQ ID NO: 3huCD3 VH CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 4 huCD3 VL CDR1 TGAVTTSNYhuCD3 VL CDR2 GTN SEQ ID NO: 5 huCD3 VL CDR3 ALWYSNLWV SEQ ID NQ: 6huCD3 VH1 EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTL VTVSS SEQ ID NO: 7 huCD3 VH2EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTL VTVSS SEQ ID NQ: 8 huCD3 VH3EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTL VTVSS SEQ ID NO: 9 huCD3 VH4EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTM VTVSS SEQ ID NO: 10 huCD3 VL1QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQA DDESIYFCALWYSNLWVFGGGTKLTVLSEQ ID NO: 11 huCD3 VL2 QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQA DDESIYFCALWYSNLWVFGGGTKLTVLSEQ ID NO: 12 huCD3 VL3 QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQA DDESDYYCALWYSNLWVFGGGTKLTVLSEQ ID NO: 13 Mature human QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNCD3ε (epsilon) DKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPN PDYEPIRKGQRDLYSGLNQRRISEQ ID NO: 14 Human CD3δ FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRI(delta) LDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQ VYQPLRDRDDAQYSHLGGNWARNKSEQ ID NO: 15 IgG1m(f) heavyASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL chain constantTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT regionKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 16IgG1m(f)-LFLEDA ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSheavy chain GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKconstant region RVEPKSCDKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTC VVV AVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 17VH huCLB-T3/4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAPGKGLEWVATISRYSRYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRPLYGSSPDYWGQGTLVTVSS SEQ ID NO: 18 VL huCLB-T3/4EIVLTQSPATLSLSPGERATLSCSASSSVTYVHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGS GTKLEMR SEQ ID NO: 19VH HER2 169 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWVRQAPGQGLEWMGWLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDRIVVRPDYFDYWGQGTLVTVSS SEQ ID NO: 20 VL HER2 169EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRT FGQGTKVEIKSEQ ID NO: 21 Mature cyno QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKE CD3ε (epsilon)DSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI SEQ ID NO: 22 Mature rhesusQDGNEEMGSITQTPYHVSISGTTVILTCSQHLGSEVQWQHNGKNKE CD3ε (epsilon)DSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI SEQ ID NO: 23 IgG1m(f)-F405LASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SF LLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 24 IgG1m(f)-K409RASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYS RLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 25 IgGlm(f)-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LFLEDA-F405LGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPE FEGGPSVFLFPPKPKDTLMISRTPEVTC VVV AVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSF LLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 26 IgGlm(f)-ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS LFLEDA-K409RGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPE FEGGPSVFLFPPKPKDTLMISRTPEVTC VVV AVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYS RLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 27 Parent murine EVKLLESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKG VHLEWVARIRSKYNNYATYYADSV KDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSA SEQ ID NO: 28 Parent murine QAVVTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDH VLLFTGLIGGTNKRAPGVPARFSG SLIGDKAALTITGAQTEDEAIY FCALWYSNLWVFGGGTKLTVLSEQ ID NO: 29 VH CD20-7D8 EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWNSGTIGYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS SEQ ID NO: 30 VL CD20-7D8EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPITFGQGTRLEIKSEQ ID NO: 31 Human GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWIgLC2/IgLC3 KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR constant domainSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 32 VL huCD3-LKNH QAVVTQEPS LSVSPGGTVTLTCRSSTGAVTTSNYANWVQQ K PGQAFRGLIGGTN NRAPGVPARFSGSLIGDKAALTITGAQ ADDESIYFCALWYSN H WVFGGGTKLTVL SEQ ID NO: 33VL huCD3-T41K QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQ KPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQ ADDESIYFCALWYSNLWVFGGGTKLTVLSEQ ID NO: 34 VH CD20-7D8 GFTFHDYA CDR1 SEQ ID NO: 35 VH CD20-7D8ISWNSGTI CDR2 SEQ ID NO: 36 VH CD20-7D8 AKDIQYGNYYYGMDV CDR3SEQ ID NO: 37 VL CD20-7D8 QSVSSY CDR1 VL CD20-7D8 DAS CDR2 SEQ ID NO: 38VL CD20-7D8 QQRSNWPIT CDR3 SEQ ID NO: 39 VH CD20-2F2EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVS S SEQ ID NO: 40 VL CD20-2F2EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPITFGQGTRLEIKSEQ ID NO: 41 VH CD20-2F2 GFTFNDYA CDR1 SEQ ID NO: 42 VH CD20-2F2ISWNSGSI CDR2 SEQ ID NO: 43 VH CD20-2F2 AKDIQYGNYYYGMDV CDR3SEQ ID NO: 44 VL CD20-2F2 QSVSSY CDR1 VL CD20-2F2 DAS CDR2 SEQ ID NO: 45VL CD20-2F2 QQRSNWPIT CDR3 SEQ ID NO: 46 VH CD20-11B8EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGLEWVSIIGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMAVYYCARDYYGAGSFYDGLYGMDVWGQGTT VTVSS SEQ ID NO: 47 VL CD20-11B8EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRSDWPLTFGGGTKVEIKSEQ ID NO: 48 VH CD20-11B8 GFTFSYHA CDR1 SEQ ID NO: 49 VH CD20-11B8IGTGGVT CDR2 SEQ ID NO: 50 VH CD20-11B8 ARDYYGAGSFYDGLYGMDV CDR3SEQ ID NO: 51 VL CD20-11B8 QSVSSY CDR1 VL CD20-11B8 DAS CDR2SEQ ID NO: 52 VL CD20-11B8 QQRSDWPLT CDR3 SEQ ID NO: 53 VH CD20-2C6AVQLVESGGGLVQPGRSLRLSCAASGFTFGDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDNQYGSGSTYGLGVWGQGTLVT VSS SEQ ID NO: 54 VL CD20-2C6EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPLTFGGGTKVEIKSEQ ID NO: 55 VH CD20-2C6 GFTFGDYT CDR1 SEQ ID NO: 56 VH CD20-2C6ISWNSGSI CDR2 SEQ ID NO: 57 VH CD20-2C6 TKDNQYGSGSTYGLGV CDR3SEQ ID NO: 58 VL CD20-2C6 QSVSSY CDR1 VL CD20-2C6 DAS CDR2 SEQ ID NO: 59VL CD20-2C6 QQRSNWPLT CDR3 SEQ ID NO: 60 VL huCD3-CDR3 ALWYSN H WV

The CDR regions have been annotated according to the IMGT definitions.

EXAMPLES Example 1 Generation of Humanized CD3 Antibodies andNon-Activating Antibody Variants Humanization of CD3 Antibodies

Humanization of a murine CD3 antibody (U.S. Pat. No. 8,236,308,described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK)using their improved version of the germline humanization (CDR-grafting)technology (EP 0 629 240). Using this technology, 4 different VH chains(SEQ ID NOs:6, 7, 8, and 9) and 3 different VL chains (SEQ ID NOs:10,11, and 12) were designed. By combining these 4 VH with the 3 VL chains,12 different antibodies were generated. The humanized variants aredescribed herein as huCD3. Thus, humanized variants comprising a VH anda VL according to the invention, are described as, e.g., IgG1-huCD3-H1L1meaning that said specific variant is of the IgG1 isotype, is ahumanized CD3 and comprises the VH amino acid sequence termed “H1” andis defined according to SEQ ID NO:6, and the VL amino acid sequencetermed “L1” and is defined according to SEQ ID NO: 10. Thus, H1 refersto the variable heavy chain region VH1, L1 refers to the variable lightchain region VL1, and so forth.

In particular, the variants IgG1-huCD3-H1L1 (humanized CD3 comprisingthe VH1 sequence set forth in SEQ ID NO:6 and the VL1 sequence set forthin SEQ ID NO:10), IgG1-huCD3-H1L2 (humanized CD3 comprising the VH1sequence set forth in SEQ ID NO:6 and the VL2 sequence set forth in SEQID NO:11), IgG1-huCD3-H1L3 (humanized CD3 comprising the VH1 sequenceset forth in SEQ ID NO:6 and the VL3 sequence set forth in SEQ IDNO:12), IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequence setforth in SEQ ID NO:8 and the VL3 sequence set forth in SEQ ID NO:12),IgG1-huCD3-H4L1 (humanized CD3 comprising the VH4 sequence set forth inSEQ ID NO:9 and the VL1 sequence set forth in SEQ ID NO:10),IgG1-huCD3-H3L1 (humanized CD3 comprising the VH3 sequence set forth inSEQ ID NO:8 and the VL1 sequence set forth in SEQ ID NO:10),IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequence set forth inSEQ ID NO:8 and the VL3 sequence set forth in SEQ ID NO:12), andIgG1-huCD3-H4L3 (humanized CD3 comprising the VH4 sequence set forth inSEQ ID NO:9 and the VL3 sequence set forth in SEQ ID NO:12) have beengenerated and tested in the herein described examples.

In some examples an antibody comprising the heavy and light chainvariable region sequences of huCLB-T3/4 (SEQ ID NOs:17 and 18,respectively) were used as a control antibody (Labrijn et al., PNAS2013, 110: 5145-50) and to verify different non-activating mutationcombinations in the Fc region (see Examples 8 to 10). The huCBL-T3/4 isa humanized version of the murine CD3 antibody CLB-T3/4 (Parren et al.,Res Immunol. 1991, 142(9):749-63). Both sequences (SEQ ID NOs:17 and 18)were cloned into the relevant pcDNA3.3 (Invitrogen) expression vectorsand expressed by cotransfection in HEK293F cells. The resulting controlantibody is described as IgG1-huCLB-T3/4.

In some examples an antibody comprising the heavy and light chainvariable region sequences of the CD20 antibody 7D8 (SEQ ID NO: 29corresponding to the VH sequence and SEQ ID NO:30 corresponding to theVL sequence) was used as a positive control. When used in the context ofa positive control it is termed “IgG1-CD20”.

These IgG1-CD3 (i.e. the chimeric, parental CD3 antibody), IgG1-huCD3and IgG1-huCLB-T3/4 antibodies were used in monospecific and bispecificformat, where the bispecific antibodies were generated as describedbelow.

HER2 Antibody

In some of the examples an antibody against HER2 was used. The VH and VLsequences for this HER2-specific antibody (antibody 169, SEQ ID NOs:19and 20, respectively) were described before (WO2012/143524 [Genmab];Labrijn et al., PNAS 2013, 110: 5145-50). The antibody was used in bothmonospecific and bispecific formats, and is termed “IgG1-HER2”.

b12 Antibody

In some of the examples the antibody b12, a gp120 specific antibody(Barbas, CF. J Mol Biol. 1993 Apr. 5; 230(3):812-23.) was used as anegative control, and is termed “IgG1-b12”.

Expression

Antibodies were expressed as IgG1,κ or IgG1,λ, with or without thenon-activating mutations described below and with a mutation in the CH3domain enabling the generation of bispecific antibodies by the methoddescribed below: IgG1-HER2-K409R, IgG1-b12-K409R, IgG1-CD3-F405L.Plasmid DNA mixtures encoding both heavy and light chain of antibodieswere transiently transfected to Freestyle HEK293F cells (Invitrogen, US)using 293fectin (Invitrogen, US) essentially as described by themanufacturer.

Purification of Antibodies

Culture supernatant was filtered over 0.2 μm dead-end filters, loaded on5 mL MabSelect SuRe columns (GE Health Care) and eluted with 0.1 Msodium citrate-NaOH, pH 3. The eluate was immediately neutralized with2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH2PO4, 140 mMNaCl, pH 7.4 (B. Braun). Alternatively, subsequent to purification, theeluate was loaded on a HiPrep Desalting column and the antibody wasexchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer.After dialysis or exchange of buffer, samples were sterile filtered over0.2 μm dead-end filters. Purity was determined by SDS-PAGE andconcentration was measured by absorbance at 280 nm. Purified antibodieswere stored at 2-8° C.

Generation of Bispecific Antibodies

Bispecific antibodies were generated in vitro using the DuoBody®platform technology, i.e. 2-MEA-induced Fab-arm exchange as described inWO 2011/147986 and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5: 962-973). To enable the productionof bispecific antibodies by this method, IgG1 molecules carrying asingle mutation in the CH3 domain were generated: in one parental IgG1antibody the F405L mutation (i.e. the IgG1-CD3 antibody), in the otherparental IgG1 antibody the K409R mutation (i.e. the HER2 or b12antibodies). To generate bispecific antibodies, these two parentalantibodies, each antibody at a final concentration of 0.5 mg/mL, wereincubated with 25 or 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a totalvolume of 500 μL TE at 31° C. for 5 hours. The reduction reaction wasstopped when the reducing agent 2-MEA is removed by using PD-10 columns(GE-healthcare, product #17-0851-01), equilibrated with 25 mL PBS. Priorto desalting, 2 mL PBS (B. Braun, product #3623140) was added to thesamples to adjust the volume to 2.5 mL. Elution was done in 3.5 mL PBS.Samples were collected in Amicon Ultra centrifugal units (30 kD MWCO,Millipore, product #UFC803096) and concentrated by centrifuging 8 min at3000×g. Volumes were adjusted to 500 μL (when needed) with PBS andsamples were sterile-filtered over a 0.2 μm filter (Millex-GV, product#SLGV004SL). The bispecific products were stored at 2-8° C.

In an alternative way, yielding the same bispecific antibody, togenerate bispecific antibodies 100 μg of the two parental antibodieswere mixed and incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) ina total volume of 400 μL PBS (B. Braun, product #3623140) at 31° C. for5 hours. The reduction reaction was stopped when the reducing agent2-MEA is removed by using Amicon Ultra 0.5 ml centrifugal units (10 kDMWCO, Millipore, product #UFC501096) and washing 4× with 400 μl PBS bycentrifuging 10 min at 13000×g. Samples were collected in a new tube byinverting the filter and centrifuging 2 min at 1000 g. Volumes wereadjusted to 200 μL (when needed) with PBS. The absorbance of 280 nm(A280) of bispecific products was measured to determine the finalconcentration. HPLC cation exchange chromatography (HPLC-CEX) (asdescribed in WO 2013/060867) was performed to determine the amount ofbispecific product. Samples were stored at 2-8° C.

The generated bispecific antibodies are described as “K409R IgG1backbone” and “F405L IgG1 backbone” in the following.

Non-Activating Mutations

Several antibody variants were generated with one or more amino acidsubstitutions in the Fc region. A non-activating Fc region prevents theantibody from interacting with Fc-receptors present on blood cells, suchas monocytes, or with C1q to activate the classical complement pathway.Reduction of the Fc activity was tested in antibody variants thatcontain different combinations of amino acid substitutions in the Fcregion. Maximally five amino acid substitutions were introduced, whichinclude the mutations N297Q, L234A, L235A, L234F, L235E, D265A, andP331S. Substitutions in one or more of these five amino acid positionswere introduced in the K409R and/or F405L IgG1 backbone. The followingFc region variants of the huCLB-T3/4 antibody were generated: N297Q(refers to the N297Q substitution, termed IgG1-huCLB-T3/4-N297Q), LFLE(refers to the L234F/L235E substitutions, termed IgG1-huCLB-T3/4-LFLE),LALA (refers to the L234A/L235A substitutions, termedIgG1-huCLB-T3/4-LALA), LFLENQ (refers to the L234F/L235E/N297Qsubstitutions, termed IgG1-huCLB-T3/4-LFLENQ), LFLEDA (refers to theL234F/L235E/D265A substitutions, termed IgG1-huCLB-T3/4-LFLEDA), DA(refers to the D265A substitution, termed IgG1-huCLB-T3/4-DA), DAPS(refers to the D265A/P331S substitutions, termed IgG1-huCLB-T3/4-DAPS),DANQ (refers to the D265A/N297Q substitutions, termedIgG1-huCLB-T3/4-DANQ), LFLEPS (refers to the L234F/L235E/P331Ssubstitutions, termed IgG1-huCLB-T3/4-LFLEPS), and LFLEDANQPS (refers tothe L234F/L235E/D265A/N297Q/P331S substitutions, termedIgG1-huCLB-T3/4-LFLEDANQPS).

In particular, in the IgG1-huCD3 antibody variants a combination ofthree amino acid substitutions, which include the mutations L234F, L235Eand D265A and is referred to as LFLEDA, were introduced in the K409R andF405L IgG1 backbones to generate antibodies with a non-activating Fcregion. The resulting non-activating antibody variant is termed with thesuffix “-LFLEDA”.

Example 2 Binding of Humanized CD3 Antibodies and Non-ActivatingVariants Thereof to Human and Cynomolgous T-Cell Lines Expressing CD3

Binding of purified variants of humanized CD3 (huCD3) antibodies andbispecific (bs)IgG1-huCD3×HER2 molecules with or without LFLEDAmutations in the Fc region (see Example 1) to the human T-cell lineJurkat (Clone E6-1, ATCC® TIB-152™, LGC Standards GmbH, Wesel, Germany)or the cynomolgous T-cell line HSC-F (cat. no. JCRB1164; Health ScienceResearch Resources Bank, Osaka, Japan) was analyzed by FACS analysis. Inaddition to the non-activating mutations, LFLEDA, the antibody variantscontained F405L or K409R mutations as described in Example 1.

Cells (1×10⁵ cells/well) were incubated in polystyrene 96-wellround-bottom plates (Greiner bio-one 650101) with serial dilutions ofantibody preparations (range 5 to 10,000 ng/mL in 3-fold dilutions) in100 μL PBS/0.1% BSA/0.02% azide at 4° C. for 30 min.

After washing twice in PBS/0.1% BSA/0.02% azide, cells were incubated in100 μL with secondary antibody at 4° C. for 30 min. As a secondaryantibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2(109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove,Pa.) diluted 1/100 in PBS/0.1% BSA/0.02% azide, was used for allexperiments. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide,re-suspended in 150 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences). Binding curves were analyzed using non-linearregression (sigmoidal dose-response with variable slope) using GraphPadPrism V 5.04 software (GraphPad Software, San Diego, Calif., USA).

FIG. 1A shows that binding to Jurkat cells of the IgG1λ-huCD3 variantsIgG1-huCD3-H1L1 (SEQ ID NOs:6 and 10, respectively), IgG1-huCD3-H1L2(SEQ ID NOs:6 and 11, respectively), IgG1-huCD3-H1L3 (SEQ ID NOs:6 and12, respectively), IgG1-huCD3-H3L3 (SEQ ID NOs:8 and 12, respectively),and IgG1-huCD3-H4L1 (SEQ ID NOs:9 and 10, respectively) with wild-typeFc region was observed for all variants, and that binding ability ofIgG1-CD3-LFLEDA (parental CD3 antibody as described in Example 1 withnon-activating LFLEDA mutations) and IgG1-huCD3-H3L1-LFLEDA withnon-activating LFLEDA mutations were similar to huCD3 variants withwild-type Fc regions. Binding of IgG1-huCLB-T3/4, included as positivecontrol, was strong to Jurkat cells in comparison with the IgG1-huCD3variants. No binding was observed for the negative control antibodyIgG1-b12.

FIG. 6A shows that binding to Jurkat cells of the IgG1-CD3-LFLEDA(parental CD3 antibody as described in Example 1 with non-activatingLFLEDA mutations), IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA,IgG1-3huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA,and IgG1-huCD3-H4L3-LFLEDA with the non-activating LFLEDA mutations weresimilar to binding ability to huCD3 variants with wild-type Fc regions.Binding of IgG1-huCLB-T3/4, included as positive control, was strongerto Jurkat cells in comparison with the IgG1-huCD3 variants in lowantibody concentrations but similar at higher antibody concentrations.Overall, the humanized CD3 variants have maintained the binding abilityto CD3 as the IgG1-CD3 antibody. No binding was observed for thenegative control antibody IgG1-b12.

FIG. 1B shows that bispecific antibody variants bsIgG1 CD3×HER2, bsIgG1CD3×b12-LFLEDA, and bsIgG1 huCD3-H3L1×HER2-LFLEDA also bind to Jurkatcells. The maximal binding values for these bispecific antibodies arehigher than the maximal binding values of the monospecific antibodies.The EC50 concentrations of the bispecific antibodies were 6 to 10-foldhigher. Again, no binding was observed for the negative control antibodyIgG1-b12.

FIG. 6B shows that bispecific non-activating Fc antibody variants bsIgG1huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA,bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA,bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA alsobind to Jurkat cells. The maximal binding values for these bispecificantibodies are higher than the maximal binding values of themonospecific antibodies. The EC50 concentrations of the bispecificantibodies were 4 to 10-fold higher. Monovalent binding allows moreantibodies to accumulate on the cell surface, thus, the higher bindingvalues for the bispecific antibodies. Again, no binding were observedfor the negative control antibody IgG1-b12.

FIG. 2A shows that binding of the IgG1-huCD3 variants IgG1-huCD3-H1L1,IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1with wild-type Fc region and IgG1-CD3-LFLEDA, IgG1-huCD3-H3L1-LFLEDA tothe cynomolgus T-cell line HSC-F was similar. No binding was observedfor the control antibody huCLB-T3/4, which does not cross-react withcynomolgus CD3, and the negative control antibody IgG1-b12.

FIG. 7A shows that binding of the IgG1-huCD3 variants IgG1-CD3-LFLEDA,IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA,IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, andIgG1-huCD3-H4L3-LFLEDA to the cynomolgus T-cell line HSC-F was similar.No binding was observed for the negative control antibody IgG1-b12.

FIG. 2B shows that bispecific antibody variants bsIgG1 CD3×HER2 andbsIgG1 huCD3-H3L1-LFLEDA also bind to HSC-F cells. The maximal bindingvalues for these bispecific antibodies are higher than the maximalbinding values of the monospecific anti-CD3 variants. The EC50concentrations of the bispecific antibodies were 10 to 12-fold higherthan that of the monospecific anti-CD3 antibodies. Again, no binding wasobserved for the negative control antibody IgG1-b12.

FIG. 7B shows that bispecific non-activating Fc antibodies variantsbsIgG1 huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA,bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA,bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA alsobind to HSC-F cells. The maximal binding values for these bispecificantibodies are higher than the maximal binding values of themonospecific anti-CD3 variants. The EC50 concentrations of thebispecific antibodies were 3 to 6-fold higher than that of themonospecific anti-huCD3 antibodies. Again, no binding was observed forthe negative control antibody IgG1-b12.

Example 3 T-Cell Activation by Humanized CD3 Antibody Variants

CD69 expression is an early marker of T-cell activation. CD3 antibodiescould mediate cross-linking of T-cells and immune cells through bindingof CD3 expressed by T-cells and Fc receptors expressed by immune cellsby the Fc region of the antibody, such as the IgG1 Fc region. This couldlead to T-cell activation and induction of CD69. Antibody variantscontaining a non-activating Fc region (LFLEDA mutations) do not bind Fcreceptors. Therefore, it was anticipated that non-activating CD3antibodies do not induce T-cell activation and CD69 expression as thenon-activating Fc region does not bind to Fc receptor expressing immunecells and thus cannot cross-link T-cells and immune cells.

CD69 expression on T-cells was evaluated by FACS analysis to determineearly activation of T-cells after incubation with humanized CD3 (huCD3)variants with and without LFLEDA mutations in the Fc region. In additionto the non-activating mutations, LFLEDA variants contain F405L or K409Rmutations as described in Example 1.

PBMCs were isolated from whole blood or buffy coat by density gradientseparation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a/dRijn, The Netherlands), washed with PBS and re-suspended in culturemedium.

A dose response series of huCD3 antibody variants, a negative control(IgG1-b12) and positive controls (IgE-huCD3 and parental IgG1-CD3) wereprepared in culture medium (ranging from 0.1 to 1,000 ng/mL in 10-folddilutions) and added to the wells of a 96-well round bottom platecontaining human or cynomolgus PBMCs. After 16-24 hours incubation,cells were pelleted by centrifugation and supernatant (containingcytokines) was collected and stored at −20° C. Cells were then washedwith PBS/0.1% BSA/0.02% azide and stained for 30 minutes at 4° C. with amouse-anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, TheNetherlands; T-cell marker) and mouse-anti-human CD69-APC antibody(340560; BD Biosciences, Franklin Lakes, N.J.), which are cross-reactivewith cynomolgus CD28 and CD69, respectively. Unbound antibodies wereremoved by washing twice with PBS/0.1% BSA/0.02% azide. Cells werere-suspended in 150 μL/well and CD69-expression on CD28 positive cellswas measured on FACS Canto II (BD Biosciences).

FIG. 3 shows that IgG1-CD3 (as described in Example 1) and humanizedIgG1-huCD3 variants with wild-type IgG1 Fc region induced similar levelsof CD69 expression on T-cells from human (FIG. 3A) and cynomolgus (FIG.3B) origin. Non-activating (LFLEDA) IgG1-CD3-LFLEDA and IgG1-huCD3-H3L1variants induced low levels of CD69 expression in human T-cells. Noexpression of CD69 was induced by the non-activating IgG1-huCD3 variantsin cynomolgus T-cells. The control antibody IgG1-b12 also did not induceexpression of CD69 in human or cynomolgus T-cells.

FIG. 8 shows that non-activating (LFLEDA) IgG1-huCD3-H3L1-LFLEDA,IgG1-huCD3-H3L3-LFLEDA, IgG1-3huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA,IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA variants induced lowlevels of CD69 expression in human T-cells. FIGS. 8A and 8B showinduction of CD69 expression on T-cells from cynomolgus monkey. Theminor activation by non-activating variants observed may be due tocross-linking of CD3 molecules through bivalent binding of CD3antibodies. Such explanation is supported by the observation that theactivation is reduced at the highest concentration where antibodybinding is monovalent. The control antibody IgG1-b12 also did not induceexpression of CD69 in human or cynomolgus T-cells.

FIGS. 8C and 8D show that non-activating bispecific antibody variantsbsIgG1-huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA,bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA,bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA do notinduce expression of CD69 in T-cells from humans (FIG. 8C) or cynomolgusmonkeys (FIG. 8D). However, at the higher antibody concentrations someinduction of CD69 expression was observed.

Example 4 T-Cell Proliferation Induced by Humanized CD3 AntibodyVariants

The effect of humanized CD3 (huCD3) antibody variants (described inExample 1) on the proliferation of human and cynomolgus T-cells wasevaluated by the Cell proliferation ELISA kit from Roche Applied Science(Cell Proliferation ELISA, BrdU kit, #11647229001; Roche AppliedScience, Mannheim, Germany), which was performed according to themanufacturer's instructions.

Human or cynomolgus PBMCs, isolated from whole blood or buffy coat, wereincubated in 96-well culture plates with dilution series (ranging from0.1 to 1,000 ng/mL in 10-fold dilutions) of IgG1 huCD3 antibodyvariants. IgE-CD3 and IgG1-huCLB-T3/4 were included as positive controlsand IgG1-b12 as negative control. After 3 days of incubation with theantibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added tothe medium and plates were incubated for 5 hours. Cells were thenpelleted by centrifugation and supernatant collected and stored at −20°C. Plates were dried and stored at 4° C. until ELISA was performed.

BrdU incorporation in the DNA was determined by ELISA according to themanufacturer's instructions (Roche Applied Science). Cells were fixed tothe plates, where after the plates were incubated for 90 minutes at RTwith an anti-BrdU antibody conjugated with peroxidase. Plates werewashed with PBST and binding was detected using ABTS buffer (instead ofthe TMB solution provided with the kit). Color development was stoppedafter 30 min by adding 2% oxalic acid to the wells. OD405 nm was thenmeasured on an EL808 ELISA-reader.

FIG. 4 shows that incubation of PBMCs with parental IgG1-CD3 andhumanized IgG1-huCD3 variants with wild-type IgG1 Fc region inducedstrong proliferation of human (FIG. 4A) and cynomolgus (FIG. 4B)T-cells, even at very low concentrations of antibody. Incubation withnon-activating LFLEDA variants of the IgG1-huCD3 antibodies did notinduce proliferation of human T-cells (FIGS. 4A and 9A) or cynomolgusT-cells (FIGS. 4B and 9B). Thus, although the non-activating variants ofthe IgG1-huCD3 antibodies induced low levels of CD69 expression in humanT-cells (as shown in Example 3), no proliferation of human T-cells wasinduced by these non-activating IgG1-huCD3 variants.

FIGS. 9C and 9D show that non-activating bispecific antibody variantsbsIgG1-huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA,bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA,bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA do notinduce proliferation of T-cells isolated from humans (FIG. 9C) orcynomolgus monkeys (FIG. 9D).

Example 5 In Vitro T-Cell-Mediated Cytotoxicity Induced by Humanized CD3Antibody Variants

Tumor-specific T-cell cytotoxicity can be mediated by bispecificantibodies that bind with one arm to CD3 and the other arm to atumor-specific target, such as HER2. Simultaneous binding of bispecificantibodies to both T-cells and tumor cells will lead to T-cellactivation and tumor cell specific cytotoxicity. In this Example, T-cellmediated cytotoxicity against HER2-positive tumor cells was evaluatedusing bispecific antibodies against CD3 (humanized variants) and HER2.

Therefore, AU565 (human breast carcinoma) cells were cultured in RPMI1640 supplemented with 10% (vol/vol) heat inactivated CCS, 1.5 g/Lsodium bicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g/L glucose(Sigma), 50 IU/mL penicillin, and 50 μg/mL streptomycin. The cell linewas maintained at 37° C. in a 5% (vol/vol) CO₂ humidified incubator.AU565 cells were cultured to near confluency, after which cells weretrypsinized, re-suspended in culture medium and passed through a cellstrainer to obtain a single cell suspension. 5×10⁴ cells were seeded ineach well of a 96-well culture plate, and cells were incubated at least3 hrs at 37° C., 5% CO₂ to allow adherence to the plate.

Human or cynomolgus PBMCs were isolated from whole blood or buffy coat.Isolated PBMCs were washed with PBS, re-suspended in culture medium andadded in a 1:1 ratio to the AU565 tumor cells in the 96-well plates. Thepercentage of T-cells present in PBMCs was measured by FACS-analysis,using a mouse anti-human CD3-PerCP (BD, #345766) antibody (for stainingT-cells), which is cross-reactive with cynomolgus CD3. The T-cellcontent in the population of used PBMCs was typically 50 to 60%.

Dilution series (final concentrations ranging from 0.001 up to 10,000ng/mL) of bispecific antibody variants bsIgG1 CD3×HER2-LFLEDA, bsIgG1CD3×b12-LFLEDA, bsIgG1 huCD3-H3L1×HER2-LFLEDA,bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA,bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, andbsIgG1-huCD3-H4L3×HER2-LFLEDA were prepared in culture medium and addedto the plates. IgG1-HER2-LFLEDA and IgG1-b12 were included as controls.In addition to the non-activating mutations, LFLEDA antibody variantscontain F405L or K409R mutations for preparation in bispecific format(see Example 1). Plates were incubated for 3 days at 37° C., 5% CO₂.Incubation of cells with 1 μM staurosporin (#S6942-200, Sigma) was usedas reference for 100% tumor cell kill. Plates were washed twice withPBS, and 150 μL culture medium containing 10% Alamar blue was added toeach well. Plates were incubated for 4 hours at 37° C., 5% CO₂.Absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham,Mass.).

Bispecific CD3×HER2-LFLEDA antibody variants(bsIgG1-huCLB-T3/4×HER2-LFLEDA and bsIgG1-CD3×HER2-LFLEDA) inducedkilling of AU565 cells at low concentrations using human effector cells(FIG. 5A) or cynomolgus effector cells (FIG. 5B). The CD3 bispecificcontrol antibody huCLB-T3/4×HER2-LFLEDA, which shows no cross-reactivitywith cynomolgus CD3, only induced killing of AU565 cells when humanPBMCs were used (FIG. 5A). Thus, no killing of target cells was observedwhen cynomolgus effector cells were used in the assay (FIG. 5B).Incubation with monospecific IGG1-b12 or IgG1-HER2-LFLEDA orbsIgG1-CD3×b12-LFLEDA antibodies did not induce unspecific killing oftarget cells.

Bispecific antibody variants bsIgG1 huCD3-H3L1×HER2-LFLEDA,bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA,bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, andbsIgG1-huCD3-H4L3×HER2-LFLEDA induced killing of AU565 cells at lowconcentrations using human effector cells (FIG. 10A) or cynomolguseffector cells (FIG. 10B). Incubation with monospecific IgG1-b12 orIgG1-HER2-LFLEDA antibodies did not induce unspecific target cellkilling (FIGS. 10A and B). Thus, the humanized CD3 variants comprisingthe non-activating Fc region do not induce unspecific target cellkilling, which indicates that the variants comprising a non-activatingFc region can be used to ensure targeted T-cell activation and thusavoid non-targeted T-cell activation.

Example 6 Rhesus T-Cell Activation by Humanized CD3 Antibody Variants

CD69 expression on rhesus T-cells was evaluated to determine earlyactivation of T-cells after incubation with humanized CD3 (huCD3)antibody variants with wild-type IgG1 Fc region. Isolation of rhesusPBMCs and evaluation of CD69 expression by flow cytometry was performedas described in Example 3.

FIG. 11 shows that humanized CD3 antibody variants IgG1-huCD3-H1L1,IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1induced CD69 expression on T-cells from rhesus origin to similar levelsas IgG1-CD3 (as described in Example 1). The negative control antibodyIgG1-b12 did not induce expression of CD69 in rhesus T-cells. Thus, thehuCD3 variants according to the present invention may be used inexperiments involving rhesus monkey CD3. The huCD3 variants arecross-reactive with rhesus monkey CD3.

Example 7 T-Cell Activation by Non-Activating Variants of huCLB-T3/4

CD69 expression on T-cells was evaluated by FACS analysis to determineearly activation of T-cells after incubation with IgG1-huCLB-T3/4variants with mutations in the Fc region (see Example 1).

PBMCs were isolated from whole blood or buffy coat by density gradientseparation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a/dRijn, The Netherlands), washed with PBS and resuspended in culturemedium.

A dose response series of IgG1-huCLB-T3/4 variants, a negative control(IgG1-huCLB-T3/4-Fab) and positive control (IgE-huCLB-T3/4) wereprepared in culture medium (ranging from 1 to 1000 ng/mL in 3-folddilutions) and added to the wells of a 96-well round bottom platecontaining the PBMCs. After 16-24 hours incubation, cells were pelletedby centrifugation and supernatant (containing cytokines) collected andstored at −20° C. Cells were then washed with PBS/0.1% BSA/0.02% azideand stained for 30 minutes at 4° C. with a mouse-anti-human CD28-PE(854.222.010; Sanquin, Amsterdam, The Netherlands; T-cell marker) andmouse-anti-human CD69-APC antibody (340560; BD Biosciences, FranklinLakes, N.J.). Unbound antibodies were removed by washing twice withPBS/0.1% BSA/0.02% azide. Cells were resuspended in 150 μL/well andCD69-expression on CD28 positive cells was measured on FACS Canto II (BDBiosciences).

FIG. 12A shows that CD69 expression was high on cells which wereincubated with IgE-huCLB-T3/4, IgG1-huCLB-T3/4, IgG1-huCLB-T3/4-DA andIgG1-huCLB-T3/4-DAPS. Incubation with IgG1-huCLB-T3/4-N297Q inducedsomewhat lower expression levels of CD69 compared to wild-typeIgG1-huCLB-T3/4, and incubation with IgG1-huCLB-T3/4-LFLE andIgG1-huCLB-T3/4-LFLEPS induced CD69 to a lesser extent. Incubation ofPBMCs with IgG1-CD3 Fab, IgG1-huCLB-T3/4-LFLEDA, IgG1-huCLB-T3/4-LFLENQ,IgG1-huCLB-T3/4-DANQ and IgG1-huCLB-T3/4-LFLEDANQPS antibodies did notinduce any expression of CD69 on T-cells.

FIG. 12B shows that CD69 expression was high on cells which wereincubated with IgE-huCLB-T3/4 and IgG1-huCLB-T3/4. Incubation withIgG1-huCLB-T3/4-LALA induced somewhat lower expression levels of CD69compared to wild-type IgG1-huCLB-T3/4, and incubation withIgG1-huCLB-T3/4-LFLEDA and IgG1-b12 (negative control) did not induceany expression of CD69 on T-cells.

Example 8 T-Cell Proliferation by Non-Activating Variants of huCLB-T3/4

The effect of huCLB-T3/4 variants (described in Example 1) on theproliferation of T-cells was evaluated by the Cell proliferation ELISAkit from Roche Applied Science (Cell Proliferation ELISA, BrdU kit,#11647229001; Roche Applied Science, Mannheim, Germany), which wasperformed according to the manufacturer's instructions.

PBMCs, isolated from whole blood or buffy coat, were incubated in96-well culture plates with dilution series (ranging from 0.1 to 1000ng/mL) of IgG1-CD3 variants. IgE-CD3 and IgG1-CD3 were included aspositive controls and IgG1-b12 (with K409R mutation for generation ofbispecific antibodies) as a negative control. After 3 days of incubationwith the antibodies, BrdU (Roche Applied Science, Mannheim, Germany) wasadded to the medium and plates were incubated for 5 hours. Cells werethen pelleted by centrifugation and supernatant collected and stored at−20° C. Plates were dried and stored at 4° C. until ELISA was performed.

BrdU incorporation in the DNA was determined by ELISA according to themanufacturer's instructions (Cell Proliferation ELISA, BrdU kit,#11647229001; Roche Applied Science). Cells were fixed to the plates,where after the plates were incubated for 90 minutes at room temperature(RT) with an anti-BrdU antibody conjugated with peroxidase. Plates werewashed with PBST and binding was detected using ABTS buffer (instead ofthe TMB solution provided with the kit). Color development was stoppedafter 30 min by adding 2% oxalic acid to the wells. OD405 nm was thenmeasured on an EL808 ELISA-reader.

FIG. 13A shows that incubation of PBMCs with IgG1-huCLB-T3/4,IgG1-huCLB-T3/4-DA and IgG1-huCLB-T3/4-DAPS induced strong proliferationof T-cells, even at very low concentrations of antibody. Incubation withIgG1-huCLB-T3/4-N297Q induced dose-dependent proliferation, which wascomparable to the IgE-huCLB-T3/4 positive control. Incubation of PBMCswith IgG1-huCLB-T3/4-Fab, IgG1-b12-N297Q, IgG1-huCLB-T3/4-LFLE,IgG1-huCLB-T3/4-LFLEDA, IgG1-huCLB-T3/4-LFLENQ, IgG1-huCLB-T3/4-LFLEPS,IgG1-huCLB-T3/4-DANQ and IgG1-huCLB-T3/4-LFLEDANQPS antibodies did notinduce proliferation of T-cells.

FIG. 13B shows that incubation of PBMCs with IgG1-huCLB-T3/4 inducedstrong proliferation of T-cells, even at very low concentrations ofantibody. Incubation with IgE-huCLB-T3/4 (positive control) andIgG1-huCLB-T3/4-LALA induced dose-dependent proliferation. Incubation ofPBMCs with IgG1-huCLB-T3/4-LFLEDA did not induce proliferation ofT-cells.

Based on the results from Examples 7 and 8, a subset of mutants thatwere considered least activating, was subjected to further analysis.

Example 9 In Vitro T-Cell-Mediated Cytotoxicity Induced byNon-Activating Antibody Variants huCLB-T3/4

AU565 (human breast carcinoma) cells were cultured in RPMI 1640supplemented with 10% (vol/vol) heat inactivated CCS, 1.5 g/L sodiumbicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g/L glucose (Sigma), 50IU/mL penicillin, and 50 μg/mL streptomycin. The cell line wasmaintained at 37° C. in a 5% (vol/vol) CO₂ humidified incubator. AU565cells were cultured to near confluency. Cells were trypsinized,re-suspended in culture medium and passed through a cell strainer toobtain a single cell suspension. 5×10⁴ cells were seeded in each well ofa 96-well culture plate, and cells were incubated at least 3 hrs. at 37°C., 5% CO2 to allow adherence to the plate.

Peripheral blood mononuclear cells (PBMC) were isolated from blood fromhealthy volunteers using Leucosep 30 mL tubes, according to themanufacturer's protocol (Greiner Bio-one). Isolated PBMCs were washedwith PBS, re-suspended in culture medium and added in a 1:1 ratio to theAU565 tumor cells in the 96-well plates. The percentage of T-cellspresent in PBMCs was measured by FACS-analysis, using a mouse anti-humanCD3-PerCP (BD, #345766) antibody (for staining T-cells). The T-cellcontent in the population of used PBMCs was typically 50 to 60%.

Dilution series (final concentrations ranging from 0.004 to 1000 ng/mL)of IgG1-b12, IgG1-huCLB-T3/4, IgG1-HER2, and bispecific huCLB-T3/4×b12and huCLB-T3/4×HER2 antibodies expressed as different Fc-variants, wildtype, N297Q, LFLE, LALA, LFLENQ, LFLEDA, DANQ, and LFLEDENQPS, wereprepared in culture medium and added to the plates. Plates wereincubated for 3 days at 37° C., 5% CO₂. Incubation of cells with 1 μMstaurosporin (#S6942-200, Sigma) was used as reference for 100% tumorcell kill. After incubation, supernatants were removed and stored at−20° C. Plates were washed twice with PBS, and 150 μL culture mediumcontaining 10% Alamar blue was added to each well. Plates were incubatedfor 4 hours at 37° C., 5% CO₂. Absorbance at 590 nm was measured(Envision, Perkin Elmer, Waltham, Mass.).

Two experiments were performed using PBMCs from different donors. In thefirst experiment Fc-variants N297Q, LFLE, LFLENQ, LFLEDA, DANQ, andLFLEDANQPS were tested (FIG. 14A-G). In the second experimentFc-variants LFLEDA and LALA were tested (FIG. 15A-C). Antibodies withwild-type Fc-domains were included in both experiments as reference.Incubation with wild-type monospecific IgG1-huCLB-T3/4 or bispecifichuCLB-T3/4×b12 antibodies induced unspecific killing of target cells(FIGS. 14A-G and 15A-C). The monospecific IgG1-huCLB-T3/4 andbsIgG1-huCLB-T3/4×b12 variants N297Q (FIG. 14A-G) and LALA (FIG. 15A-C)still induced some unspecific target cell killing, albeit to a lesserextent than the wild-type antibody tested in the same experiment.Unspecific target cell killing was not induced by any of the othertested IgG1-huCLB-T3/4 or bsIgG1-huCLB-T3/4×b12 antibodies withnon-activating mutations (FIGS. 14A-G and 15A-C).

All bispecific huCLB-T3/4×HER2 antibodies induced dose-dependent killingof AU565 cells with at least comparable efficacy compared to the wildtype bispecific huCLB-T3/4×HER2 antibody without non-activatingmutations (FIGS. 14A-G and 15A-C). Maximum killing occurred at very lowconcentrations.

No cytotoxicity was induced by wild-type or non-activating variants ofthe monospecific b12 or HER2 antibodies (FIGS. 14A-G and 15A-C), whichwas as expected.

Example 10 Evaluation of Binding of C1q to Non-Activating AntibodyVariants of huCLB-T3/4

Interaction of C1q with antibodies bound to a target cell is the firststep in the classical pathway of complement activation. Since wild-typeIgG1 harbors the interaction site for C1q, the interaction of C1q tothese non-activating IgG1 variants by an ELISA was evaluated.

Dilution series (range 7-30,000 ng/mL in 4-fold dilutions) ofIgG1-huCLB-T3/4, bsIgG1-huCLB-T3/4×HER2 and IgG1-CD20 (positive control)and non-activating antibody variants as described above in Example 1thereof were coated on 96-well Microlon ELISA plates (Greiner, Germany)overnight at 4° C. Plates were washed and blocked with PBS supplementedwith 0.025% Tween 20 and 0.1% gelatine. With washings in betweenincubations, plates were sequentially incubated with 3% pooled humanserum (Sanquin, product #M0008) for 1 h at 37° C., with 100 μL/wellrabbit anti-human C1q (DAKO, product #A0136, 1/4,000) for 1 h at RT, andwith 100 μL/well swine anti-rabbit IgG-HRP (DAKO, P0399, 1:10,000) asdetecting antibody for 1 h at RT. Detection was performed by addition of1 mg/mL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS;Roche, Mannheim, Germany) for about 30 min. The reaction was stopped bythe addition of 100 μL 2% oxalic acid. Absorbance was measured at 405 nmin a microplate reader (Biotek, Winooski, Vt.). Log transformed datawere analyzed by fitting sigmoidal dose-response curves with variableslope using GraphPad Prism software.

FIG. 16A shows that the antibodies with wild-type IgG1 Fc regions,IgG1-CD20 and IgG1-huCLB-T3/4 showed C1q binding. No binding of C1q wasdetected on all evaluated antibody variants with non-activatingmutations (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS,LFLEDANQPS, LALA).

FIG. 16B shows that the antibody with wild-type IgG1 Fc reigonbsIgG1-huCLB-T3/4×HER2 showed C1q binding. No binding of C1q wasdetected on all evaluated antibody variants with non-activatingmutations (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS,LFLEDANQPS, LALA).

FIG. 16C and FIG. 16D show that the antibodies with wild-type IgG1 Fcregions, IgG1-CD20, IgG1-huCLB-T3/4, and bsIgG1-huCLB-T3/4×HER2 showedC1q binding. No binding of C1q was detected on the antibody variantswith non-activating mutations (LFLEDA and LALA).

Example 11 Pharmacokinetic (PK) Analysis of Non-Activating AntibodyVariants

The mice in this study were housed in a barrier unit of the CentralLaboratory Animal Facility (Utrecht, The Netherlands) and kept infilter-top cages with water and food provided ad libitum. Allexperiments were approved by the Utrecht University animal ethicscommittee. 7-10 Weeks old C.B-17 SCID mice(C.B-17/Icr-Prkdc<Scid>/IcrIcoCrl, Charles-River) were injectedintravenously with 100 μg wild-type antibody (IgG1-huCLB-T3/4,IgG1-HER2, or bsIgG-huCLB-T3/4×HER2) or non-activating variants thereof(LALA, LFLEDA, LFLENQ, DANQ or LFLEDANQPS) using 3 mice per group. 50 μLblood samples were collected from the saphenous vein at 10 minutes, 4hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibodyadministration. Blood was collected into heparin containing vials andcentrifuged for 5 minutes at 10,000×g. Plasma was stored at −20° C.until determination of antibody concentrations.

Human IgG concentrations were determined using a total hIgG sandwichELISA. For this assay, mouse mAb anti-human IgG-kappa clone MH16(#M1268, CLB Sanquin, The Netherlands), coated to 96-well Microlon ELISAplates (Greiner, Germany) at a concentration of 2 μg/mL was used ascapturing antibody. After blocking plates with PBS supplemented with0.2% bovine serum albumin, samples were added, serially diluted withELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serumalbumin), and incubated on a plate shaker for 1 h at room temperature(RT). Plates were subsequently incubated with goat anti-human IgGimmunoglobulin (#109-035-098, Jackson, West Grace, Pa.) and developedwith 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS;Roche, Mannheim, Germany). The reaction was stopped after 30 min byadding 2% oxalic acid to the wells. Absorbance was measured in amicroplate reader (Biotek, Winooski, Vt.) at 405 nm.

Plasma clearance rates (mL/day/kg) were calculated based on the areaunder the curve (AUC), according to the following equation:

${{Plasma}{\mspace{11mu} \;}{clearance}} = \frac{{Dose}\left( {µ\; g\text{/}{kg}} \right)}{A\; U\; {C\left( {µ\; g\text{/}{mL}\text{/}{day}} \right)}}$

Data analysis was performed using Graphpad prism software.

FIG. 17A shows that the plasma human IgG concentrations were lower forantibody variants N297Q, DANQ, LFLENQ, and LFLEDANQPS when compared towild-type antibodies, suggesting a faster clearance. The human IgGconcentrations in plasma for antibody variants LFLEDA and LALA weresimilar to those of wild-type antibodies.

FIG. 17B shows that the plasma clearance rates of antibody variantsN297Q, DANQ and LFLENQ were 2 to 3-fold higher than that of wild-typeantibody. The clearance rate of antibody variant LFLEDANQPS was 3-5times higher than that of wild-type antibody. Plasma clearance rates ofantibody variants LFLEDA and LALA were similar to that of wild-typeantibody.

Example 12 In Vitro Immunogenicity Assessment of the IgG1-LFLEDABackbone

In order to determine the potential for clinical immunogenicity of theIgG1-LFLEDA-K409R backbone, Antitope's EpiScreen™ platform was appliedto IgG1-HER2-LFLEDA. In short, PBMCs were isolated from a cohort of 50HLA-typed healthy donors representing the European and North Americanpopulation. After CD8+ T-cell depletion the PBMC preparations wereindividually frozen and stored. Thawed PBMCs were subsequently culturedand incubated with IgG1-HER2-LFLEDA-K409R or one of the control samples(IgG1-HER2 or IgG1-HER2-LFLE-K409R) for 5 to 8 days. The ability of thesamples to induce CD4+ T-cells responses was assessed by measuring cellproliferation ([3^(H)]-Thymidine incorporation) and IL-2 production(ELISpot assay). Donors showing responses with a stimulation index (SI;signal/baseline signal)≧1.9 in both assays were considered positive.

EpiScreen™ analysis showed that for IgG1-HER2-LFLEDA, 4 donors (8%)showed positive CD4+ T-cell responses, which was comparable to 4 (8%)and 3 (6%) positive responses for IgG1-HER2 and IgG1-HER2-LFLE,respectively (FIG. 18). Thus, IgG1-HER2-LFLEDA-K409R (as well asIgG1-HER2 and IgG1-HER2-LFLE-K409R) showed low potential forimmunogenicity with frequencies of responses below 10%. The positivecontrol humanized A33 (e.g. [84]) was used as clinical benchmark controlantibody that shows high levels of immunogenicity in the clinic androutinely induces 20-30% T-cell responses in the EpiScreen Assay.

Example 13 Generation of Mutants to Optimize the Production of theHumanized CD3 Antibodies

Generation of huCD3-L1 Mutant Plasmids

Several IgG1-huCD3-L1 LC mutants were generated in order to improve theexpression levels of IgG1-huCD3-H1L1 in transient transfection assays,cf. Table 2. The selection of residues was based on comparisons withgermline sequences or screening for the presence of rare residues inhuCD3-L1 in combination with crystal structures from homologousantibodies. The selected sequences were synthesized at GeneArt (LifeTechnologies, Germany). p33L encodes the constant domain of the humanIgLC2/IgLC3 lambda light chain of SEQ ID NO:31. p33G1f encodes theIgG1m(f) heavy chain constant region of SEQ ID NO:15.

TABLE 2 Antibody name after co-expression LC constructs LC Mutants withHC VH1 encoding plasmid p33L-huCD3-VL1 — IgG1-huCD3-H1L1p33L-huCD3-VL1-F10L F10L IgG1-huCD3-H1L1-LF10L p33L-huCD3-VL1-R23A R23AIgG1-huCD3-H1L1-LR23A p33L-huCD3-VL1-A35P A35P IgG1-huCD3-H1L1-LA35Pp33L-huCD3-VL1-T41K T41K IgG1-huCD3-H1L1-LT41K p33L-huCD3-VL1-K55N K55NIgG1-huCD3-H1L1-LK55N p33L-huCD3-VL1-L97H L97H IgG1-huCD3-H1L1-LL97Hp33L-huCD3-VL1-LKNH F10L, T41K, K55N, L97H IgG1-huCDS-H1L1-LLKNHp33L-huCD3-VL1-LTGPEAEY F10L, R47T, D71G, A82P,IgG1-huCD3-H1L1-LLTGPEAEY D83E, S86A, I87E, F89Yp33L-huCD3-VL1-LAPTGPEAEY F10L, R23A, A35P, R47T, D71G,IgG1-huCD3-H1L1-LLAPTGPEAEY A82P, D83E, S86A, I87E, F89Y

Transient Expression in Expi293F Cells

For a single antibody, the plasmids encoding heavy chain (HC) and lightchain (LC) were transiently transfected in Freestyle Expi293F cells(Life technologies, USA) using ExpiFectamine 293 (Life technologies). Intotal 1.5 μg HC encoding plasmid and 1.5 μg LC encoding plasmid (Table2) were diluted in 150 μL Opti-MEM (Gibco, USA). To prepare thetransfection mix, 8 μL ExpiFectamine 293 was diluted in 150 μL Opti-MEMand incubated for 5 minutes at room temperature. Next, the DNA/Opti-MEMand ExpiFectamine 293/Opti-MEM solutions were mixed, incubated for 20minutes at room temperature and added to 2.55 mL Expi293 ExpressionMedium containing 7.5×10⁶ Expi293F cells and 50 U/mL Pen-Strep. Thecells were incubated at 37° C., 8% CO2 and shaken at 200 rpm. To enhanceexpression, 21 hours after transfection, 15 μL enhancer mix 1 and 150 μLenhancer mix 2 were added. The cells were incubated for 4 days followedby the harvest of the supernatant. Supernatants were spun at 3,000×g andfilter sterilized over a 0.2 μm filter. The IgG expression levels weremeasured on the Octet RED (ForteBio, US) using anti-human IgG sensors(ForteBio, USA).

IgG Concentration Analysis

For all mutants listed the IgG expression levels were measuredindividually or in combination. FIG. 20 shows the measured IgGexpression level in supernatant for the panel of expressed antibodieslisted in Table 2. The IgG1-huCD3-H1L1 antibody expressed to 72 μg/mL. A4-fold increase in IgG expression was observed for theIgG1-huCD3-H1L1-LT41K mutant (295 μg/mL). Similar expression levels wereobserved for IgG1-huCD3-H1L1-LLKNH mutant (311 μg/mL) including the T41Kmutation amongst other mutations. The other mutations in theseconstructs did not show expression enhancement when tested individually(IgG1-huCD3-H1L1-LF10L, IgG1-huCD3-H1L1-LK55N, IgG1-huCD3-H1L1-LL97H)compared to IgG1-huCD3-H1L1.

A second set of expression enhancing mutations was observed for thecombination of R23A and A35P. While IgG1-huCD3-H1L1-LLTGPEAEY formatlacking R23A and A35P did not show enhanced expression (83 μg/mL),IgG1-huCD3-H1L1-LLAPTGPEAEY containing the additional R23A and A35Pmutations did show a 3-fold increase in expression (237 μg/mL).Individually, R23A or A35P did not show enhanced expression levels (56and 81 μg/mL, respectively).

Example 14 Binding of huCD3-H1L1-LFLEDA Variants to Jurkat Cells

The apparent binding affinities of the huCD3-H1L1-LFLEDA variants toJurkat cells were tested. IgG1-CD3-hmAb286 disclosed in US2012038219 wasused as a reference antibody.

Jurkat cells (DSMZ, Brunswick, Germany) were thawed at 37° C. and washedonce in 30 mL PBS and spun for 10 minutes at 300×g at 4° C. Cell pelletwas resuspended in 25 mL of PBS/0.1% BSA/0.02% azide at a finalconcentration of 0.8×10⁶ cells/mL. 100 μL of this cell suspension(0.8×10⁵ cells/well) was transferred into polystyrene 96-wellround-bottom plates (Greiner Bio-one, Alphen a/d Rijn, The Netherlands)and incubated with serial dilutions of supernatant preparations (range 3to 10,000 ng/mL in 3-fold dilutions) in PBS/0.1% BSA/0.02% azide at 4°C. for 30 minutes. After washing three times in PBS/0.1% BSA/0.02%azide, Jurkat cells were incubated in 50 μL with secondary antibody at4° C. for 30 min. As a secondary antibody, R-Phycoerythrin(PE)-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) diluted 1/200 in PBS/0.1% BSA/0.02%azide, was used for all experiments. Next, Jurkat cells were washedtwice in PBS/0.1% BSA/0.02% azide, re-suspended in 150 μL PBS/0.1%BSA/0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Bindingcurves were analyzed using nonlinear regression (sigmoidal dose-responsewith variable slope) using GraphPad Prism V 5.04 software (GraphPadSoftware, San Diego, Calif., USA).

FIG. 21 shows the binding curves, and Table 3 shows the EC₅₀ values ofthe IgG1-huCD3-H1L1-LFLEDA mutants. The T41K mutation had no effect onbinding affinity whereas the K55N and LKNH mutants showed a 5-foldlowerapparent affinity in comparison to wild type IgG1-huCD3-H1L1-LFLEDA. Theaffinity of the reference antibody IgG1-CD3-hmAb286-LFLEDA was lower incomparison to IgG1-huCD3-H1L1-LFLEDA, and was similar to theIgG1-huCD3-H1L1-LFLEDA variants containing the K55N mutation.

TABLE 3 Antibodies EC₅₀ (μg/mL) IgG1-huCD3-H1L1-LFLEDA 0.096IgG1-huCD3-H1-LFLEDA-LF10L 0.169 IgG1-huCD3-H1-LFLEDA-LT41K 0.096IgG1-huCD3-H1-LFLEDA-LK55N 0.531 IgG1-huCD3-H1-LFLEDA-LL97H 0.095IgG1-huCD3-H1-LFLEDA-LLKNH 0.438 IgG1-CD3-hmAb286-LFLEDA 0.397

Example 15 Binding of Bispecific huCD3×HER2 Affinity Variants on T Cells

Binding of purified affinity variants of bispecific (bs)IgG1-huCD3×HER2molecules with or without LFLEDA mutations in the Fc region to the humanT-cells isolated from a buffy coat (donor no: N001814268806, purchasedfrom Sanquin, Amsterdam, The Netherlands) was analyzed by FACS analysis.T cells were isolated via StemSep™ Human T cell Enrichment Kit (StemCell Technologies, Vancouver, Canada) according to provider'sinstructions. Herceptin-LbH1-mutations were identified from publisheddata from Bostrom et al. (85, 86) and had lower affinity to HER2 incomparison to Herceptin (trastuzumab).

To assess binding, isolated T cells (1×10⁵ cells/well) were incubated inpolystyrene 96-well round-bottom plates (Greiner bio-one 650101) withserial dilutions of antibody preparations (range 6.4 to 10,000 ng/mL in5-fold dilutions) in 100 μL PBS/0.1% BSA/0.02% azide at 4° C. for 30minutes. After washing twice in PBS/0.1% BSA/0.02% azide, T cells wereincubated in 50 μL with secondary antibody at 4° C. for 30 minutes. As asecondary antibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgGF(ab′)2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.)diluted 1/200 in PBS/0.1% BSA/0.02% azide, was used for all experiments.Next, T cells were washed twice in PBS/0.1% BSA/0.02% azide,re-suspended in 100 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences). Binding curves were analyzed using nonlinearregression (sigmoidal dose-response with variable slope) using GraphPadPrism V 5.04 software (GraphPad Software, San Diego, Calif., USA).

The binding curves results are shown in FIG. 22, and the EC₅₀ values areshown in Table 4. As can be seen the maximal difference between EC₅₀values of the CD3 variants is a factor 2.

TABLE 4 Antibodies EC₅₀ (μg/mL) bsIgG1-huCD3-H1L1/Herceptin-LFLEDA ~0.40bsIgG1-huCD3-H1L1/Herceptin-LbH1 -LFLEDA ~0.28bsIgG1-huCD3-H1L1/HER2-LFLEDA 0.26bsIgG1-huCD3-H1L1-LK55N/Herceptin-LFLEDA ~0.44bsIgG1-huCD3-H1L1-LK55N/Herceptin-LbH1 - 0.40 LFLEDAbsIgG1-huCD3-H1L1-LK55N/HER2-LFLEDA 0.61bsIgG1-huCD3-hmAb286/Herceptin-LFLEDA 0.53bsIgG1-huCD3-hmAb286/Herceptin-LbH1 -LFLEDA 0.68bsIgG1-huCD3-hmAb286/HER2-LFLEDA 0.46

Example 16 In Vitro T-Cell-Mediated Cytotoxicity Induced by huCD3Antibody Affinity Variants

In this Example, T-cell mediated cytotoxicity against HER2-positivetumor cells was evaluated using bispecific antibodies with differentaffinities against huCD3 and HER2. The bispecific antibodies wereprepared as described above, and contained the F405L mutation in the CD3arm, and the K409R mutation in the HER2 arm. A431 and AU565 (humanbreast carcinoma) cells with different HER2 expression levels were usedin this assay. HER2 copy numbers are ˜20,000 for A431 cells and˜1,000,000 for AU565 cells. AU565 cells were cultured in RPMI 1640 withHEPES and L-glutamine (Lonza, Basel Switzerland) supplemented with 10%(vol/vol) bovine serum with iron (Life Technologies, Germany), 1.5 g/Lsodium bicarbonate (Lonza, Germany), 1 mM sodium pyruvate (Lonza,Germany), 4.5 g/L glucose (Sigma), 50 IU/mL penicillin, and 50 μg/mLstreptomycin (Lonza, Basel, Switzerland). A431 (ATCC, epidermoidcarcinoma) cells were cultured in RPMI 1640 with HEPES and L-glutamine(Lonza, Basel, Switzerland) supplemented with 10% (vol/vol) bovine serumwith iron (Life Technologies, Germany), 50 IU/mL penicillin, and 50μg/mL streptomycin (Lonza, Basel, Switzerland). The cell lines weremaintained at 37° C. in a 5% (vol/vol) CO2 humidified incubator. AU565and A431 cells were cultured to near confluency, after which cells weretrypsinized, re-suspended in culture medium and passed through a cellstrainer to obtain a single cell suspension. AU565 and A431 cells wereseeded 3×10⁴ and 1.6×10⁴ cells/well, respectively in 96-well cultureplates, and incubated at least 3 hours at 37° C., 5% CO2 to allowadherence to the plate.

Human T cells were isolated from a buffy coat as mentioned in Example15. Isolated T cells were washed with PBS, re-suspended in culturemedium (RPMI 1640, 10% serum) and added in 1:3 ratio to the AU565 or1:10 ratio to A431 cells in the 96-well plates.

Dilution series (final concentrations ranging from 0.1 up to 10,000ng/mL) of bispecific antibody variants bsIgG1-huCD3/Herceptin-LFLEDA,bsIgG1-huCD3/Herceptin-LbH1-LFLEDA, bsIgG1-huCD3-H1L1/HER2-LFLEDA,bisIgG1-huCD3-LK55N/Herceptin-LbH1-LFLEDA,bsIgG1-huCD3-LK55N/HER2-LFLEDA, bsIgG1-huCD3-hmAb286/Herceptin-LFLEDA,bsIgG1-huCD3-hmAb286/Herceptin-LbH1-LFLEDA,bsIgG1-CD3-hmAb286/HER2-LFLEDA and bsIgG1-huCD3-H1L1/b12-LFLEDA wereprepared in culture medium and added to the plates. Plates wereincubated for 3 days at 37° C., 5% CO2. Incubation of cells with 1 μMstaurosporine (Sigma) was used as reference for 100% tumor cell kill.Plates were washed twice with PBS, and 150 μL culture medium containing10% Alamar blue (Life Technologies, Germany) was added to each well.Plates were incubated for 4 hours at 37° C., 5% CO2. Absorbance at 590nm was measured (Envision, Perkin Elmer, Waltham, Mass.).

FIG. 23 shows the viability of A431 cells (FIG. 23A) and AU565 cells(FIG. 23B) treated with CD3 and HER2 affinity variants. Lower HER2affinity in combination with high and low CD3 affinity results in lowerefficacy in both cell lines (shown as open symbols in both graphs). Thebispecific antibodies with a similar high affinity to HER2 (Herceptinand HER2) in combination with wild type huCD3-H1L1, huCD3-LK55N orCD3-hmAb286 CD3 arm showed similar efficacy on A431 cells. The affinityvariation in the CD3 arm had no effect on the efficacy on A431 cells.The bispecific antibodies with a similar high affinity to HER2(Herceptin and HER2) in combination with CD3-hmAb286 CD3 arm, thehuCD3-LK55N arm and wild type huCD3-H1L1 arm showed similar efficacieson AU565 cells with the CD3 hmAb286 CD3 arm showing a relatively higherefficacy. A lower binding affinity for the CD3 arm was more beneficialin AU565 cells which had a higher HER2 expression compared to the A431cells.

Example 17 In Vivo Tumor Killing Effect of huCD3-H1L1×HER2-LFLEDA andhuCD3-H1 L1×CD20-LFLEDA in NOD-SCID Mice

The in vivo anti-tumor efficacy of the bispecific CD3×HER2 antibodybsIgG1-huCD3-H1L1×HER2-LFLEDA was evaluated in a subcutaneous NCI-N87xenograft model. In this model human, unstimulated PBMCs, as a source ofhuman T cells, were co-inoculated with tumor cells, analogous to themodel described by Brischwein et al., (Mol. Immunol. 43 (2006),1129-1143). In these experiments six to eleven weeks old female NOD-SCID(NOD.CB17-Prkdcscid/NcrCrl) mice were used. Human PBMCs from healthydonors were isolated from a buffy coat as described in Example 15. Atday 0, a mixture containing 5×10⁶ cells of both PBMCs and NCI-N87 cellswere inoculated subcutaneously in 200 μL in the right flank of eachmouse. Within one hour of injection, the mice were randomly assigned toseven different treatment groups (n=4 per donor). Each group wasinjected intravenously (i.v.) with a single dose of (bispecific)antibody. Treatment groups are shown in Table 5. Tumor growth wasevaluated twice per week using caliper (PLEXX) until an endpoint tumorvolume of 1500 mm³, until tumors showed ulcerations or until the end ofthe study.

The results are shown in FIG. 24. As can be seen from FIG. 24A,huCD3-H1L1×HER2-LFLEDA efficiently reduced the tumor size at all threetested concentrations. FIG. 24B shows the tumor size 29 days after tumorinoculation. Tumor formation was significantly inhibited (p<0.0001,Tukeys multiple comparison test) in mice treated withbsIgG1-huCD3-H1L1×HER2-LFLEDA at doses of 0.05 mg/kg, 0.5 mg/kg and 5mg/kg compared to the PBS control group, whereas treatment with 0.005mg/kg did not affect tumor formation. Treatment with 5 mg/kg of thecontrol antibody bsIgG1-huCD3-H1L1×b12-LFLEDA did not inhibit tumorformation, whereas treatment with bsIgG1-b12×HER2-LFLEDA seemed toinduce some tumor growth inhibition compared to the PBS control, butthis was not significant according to the One-Way Anova, Tukey'smultiple comparison test.

TABLE 5 Group Antibody Dose 1 PBS 2 bsIgG1- huCD3-H1L1xHER2-LFLEDA 0.1μg (=0.005 mg/kg) 3 bsIgG1- huCD3-H1L1xHER2-LFLEDA 1 μg (=0.05 mg/kg) 4bsIgG1- huCD3-H1L1xHER2-LFLEDA 10 μg (=0.5 mg/kg) 5 bsIgG1-huCD3-H1L1xHER2-LFLEDA 100 μg (=5 mg/kg) 6 bsIgG1- huCD3-H1L1xb12-LFLEDA100 μg (=5 mg/kg) 7 bsIgG1- b12xHER2-LFLEDA 100 μg (=5 mg/kg)

The in vivo anti-tumor efficacy of bispecific CD20×CD3 antibodyhuCD3-H1L1×CD20-LFLEDA was evaluated in a subcutaneous Raji xenograftmodel. In this model human, unstimulated PBMCs, as a source of human Tcells, were co-inoculated with tumor cells, analogous to the modeldescribed by Brischwein et al., (Mol. Immunol. 43 (2006), 1129-1143).Six to eleven weeks old female NOD-SCID (NOD.CB17-Prkdcscid/NcrCrl) micewere used. Human PBMC from healthy donors were isolated from a buffycoat, washed and resuspended with PBS-0.1% BSA. At day 0, a mixturecontaining 5×10⁶ cells of both PBMCs and Raji cells were inoculatedsubcutaneously in 200 μL in the right flank of each mouse. Within onehour of injection, the mice were sorted into seven groups (n=4 perdonor) and each group was injected intravenously (i.v.) with a singledose of (bispecific) antibody. Treatment groups are shown in Table 6.Tumor growth was evaluated twice per week using caliper (PLEXX) until anendpoint tumor volume of 1500 mm³, until tumors showed ulcerations oruntil the end of the study.

The results are shown in FIG. 25. As can be seen from FIG. 25A,huCD3-H1L1×CD20-LFLEDA efficiently reduced the tumor size at the twohigher tested doses (0.5 mg/kg and 0.05 mg/kg) ofhuCD3-H1L1×CD20-LFLEDA.

In FIG. 25B, the tumor size of the individual mice are indicated at thelast day the groups were intact (day 21). Tumor formation issignificantly inhibited in mice treated with huCD3-H1L1×CD20-LFLEDA atdoses of 0.05 mg/kg and 0.5 mg/kg compared to the PBS control group(p-values were p<0.05 and p<0.01, respectively, when assessed with aOne-Way Anova, Tukey's multiple comparison test). Treatment with 0.005mg/kg did not affect tumor formation. Treatment with 0.5 mg/kg and 0.05of the control antibody b12×CD20 (bsIgG1-b12×CD20-LFLEDA) did notsignificantly inhibit tumor formation compared to the PBS control(One-Way Anova, Tukey's multiple comparison test).

TABLE 6 Group Antibody Dose 1 PBS 2 bsIgG1- huCD3-H1L1xCD20-LFLEDA 0.1μg (=0.005 mg/kg) 3 bsIgG1- huCD3-H1L1xCD20-LFLEDA 1 μg (=0.05 mg/kg) 4bsIgG1- huCD3-H1L1xCD20-LFLEDA 10 μg (=0.5 mg/kg) 5 bsIgG1-b12xCD20-LFLEDA 1 μg (=0.05 mg/kg) 6 bsIgG1- b12xCD20-LFLEDA 10 μg (=0.5mg/kg)

LIST OF REFERENCES

-   [1] Xu et al., 2000, Cell Immunol. 200(1):16-26-   [2] Herold et al., 2005, Diabetes, 54(6):1763-9-   [3] Staerz, et. al., 1985, Nature 314:628-631-   [4] Muller and Kontermann, 2010, BioDrugs 24: 89-98-   [5] Lum and Thakur, 2011, BioDrugs 25: 365-379-   [6] Linke et al., 2010, MAbs 2: 129-136-   [7] Ruf et al., 2010, Br J Clin Pharmacol 69: 617-625-   [8] Bokemeyer et al., 2009, J Clin Oncol (Meeting Abstracts), 3036-   [9] Heiss et al., 2010, Int J Cancer 127: 2209-2221-   [10] Jones et al., 2009, Lancet Oncol 10:1179-1187-   [11] Kiewe et al., 2006, Clin Cancer Res 12:3085-3091-   [12] WO 2012/162067-   [13] WO 2008/119567-   [14] Fundamental Immunology Ch. 7, Paul, W., ed., 2nd ed. Raven    Press, N.Y. (1989)-   [15] Lefranc M P, et al., 2003, Dev Comp Immunol. January;    27(1):55-77-   [16] Brochet, X. et al., 2008, Nucl. Acids Res. 36, W503-508-   [17] Giudicelli, V., Brochet, X, Lefranc, M.-P., 2011, Cold Spring    Harb Protoc. Jun. 1; 2011(6)-   [18] Sambrook et al., 1989, Molecular Cloning: A laboratory Manual,    New York: Cold Spring Harbor Laboratory Press, Ch. 15-   [19] WO92/22653-   [20] EP 0 629 240-   [21] E. Meyers and W. Miller, 1988, Comput. Appl. Biosci 4, 11-17-   [22] Needleman and Wunsch, 1970, J. Mol. Biol. 48, 444-453-   [23] Clustal W algorithm, Thompson, 1994-   [24] T cell Epitope Database from e.g. http://www.iedb.org/-   [25] Perry et al., 2008 Drugs R D 9 (6):385-396-   [26] Bryson et al., 2010, Biodrugs 24 (1):1-8-   [27] Kabat, E. A. et al., 1991, Sequences of proteins of    immunological interest. 5th Edition—US Department of Health and    Human Services, NIH publication No. 91-3242, pp 662, 680, 689-   [28] Oganesyan et al., 2008, Acta Cryst. (D64):700-4-   [29] Canfield et al., 1991, J. Exp. Med. (173):1483-91-   [30] Duncan et al., 1988, Nature (332):738-40-   [31] Shields et al., 2001, J. Biol. Chem. (276):6591-604-   [32] Idusogie E E, et al., 2000, J Immunol. 164: 4178-84-   [33] Leabman et al., 2013, MAbs; 5(6):896-903-   [34] Parren et al., 1992, J. Clin Invest. 90: 1537-1546-   [35] Bruhns et al., 2009, Blood 113: 3716-3725-   [36] WO 2011/066501-   [37] Lightle, S., et al., 2010, Protein Science (19):753-62-   [38] Brekke et al., 2006, J Immunol 177:1129-1138-   [39] Dall'Acqua W F, et al., 2006, J Immunol 177:1129-1138-   [40] Wu et al., 2010, Generation and Characterization of a Dual    Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody    Engineering, Springer Berlin Heidelberg-   [41] WO 2011/131746-   [42] WO 2002/020039-   [43] WO 98/050431-   [44] WO 2011/117329-   [45] EP 1 870 459-   [46] WO 2009/089004-   [47] US 2010 00155133-   [48] WO 2010/129304-   [49] WO 2007/110205-   [50] WO 2010/015792-   [51] WO 2011/143545-   [52] WO 2012/058768-   [53] WO 2011/028952-   [54] WO 2008/003116-   [55] U.S. Pat. No. 7,262,028-   [56] U.S. Pat. No. 7,612,181-   [57] WO 2010/0226923-   [58] U.S. Pat. No. 7,951,918-   [59] CN 102250246-   [60] WO 2012/025525-   [61] WO 2012/025530-   [62] WO 2008/157379-   [63] WO 2010/080538-   [64] Sykes and Johnston, 1997, Nat Biotech 17, 355-59-   [65] U.S. Pat. No. 6,077,835-   [66] WO 2000/70087-   [67] Schakowski et al., 2001, Mol Ther 3, 793-800-   [68] WO 2000/46147-   [69] Benvenisty and Reshef, 1986, PNAS USA 83, 9551-55-   [70] Wigler et al., 1978, Cell 14, 725-   [71] Coraro and Pearson, 1981, Somatic Cell Genetics 7, 603-   [72] U.S. Pat. No. 5,589,466-   [73] U.S. Pat. No. 5,973,972-   [74] Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509-   [75] F. Ausubel et al., 1987, ed. Current Protocols in Molecular    Biology, Greene Publishing and Wiley InterScience New York-   [76] Grant et al., 1987, Methods in Enzymol 153, 516-544-   [77] Remington: The Science and Practice of Pharmacy, 19th Edition,    Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995-   [78] Sustained and Controlled Release Drug Delivery Systems, J. R.    Robinson, ed., Marcel Dekker, Inc., New York, 1978-   [79] Srivastava (ed.), Radiolabeled Monoclonal Antibodies For    Imaging And Therapy, Plenum Press 1988-   [80] Chase, “Medical Applications of Radioisotopes,” in Remington's    Pharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp.    624-652, Mack Publishing Co., 1990-   [81] Brown, “Clinical Use of Monoclonal Antibodies,” in    Biotechnology And Pharmacy 227-49, Pezzuto et al., (eds.), Chapman &    Hall 1993-   [82] U.S. Pat. No. 5,057,313-   [83] U.S. Pat. No. 6,331,175-   [84] Ritter G, et al.; 2001, Cancer Res., 61:6851-9-   [85] Bostrom et al, Science. 2009; 323(5921):1610-4-   [86] Bostrom et al, PLosOne. 2011; 6(4)e17887

1. A humanized or chimeric antibody binding to human CD3, wherein saidantibody comprises a binding region comprising heavy chain variable (VH)region CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ IDNOs: 1, 2, and 3, respectively, and light chain variable (VL) regionCDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID NO: 4,the sequence GTN, and the sequence as set forth in SEQ ID NO: 5 or SEQID NO:60, respectively.
 2. The antibody according to claim 1, whereinsaid VH region has at least 90%, at least 95%, at least 97%, or at least99% amino acid sequence identity to the amino acid sequence as set forthin the VH sequences selected from the group consisting of; a) a VHsequence as set forth in SEQ ID NO:6; b) a VH sequence as set forth inSEQ ID NO:8; c) a VH sequence as set forth in SEQ ID NO:7; and d) a VHsequence as set forth in SEQ ID NO:9.
 3. The antibody according to anyone of the preceding claims, wherein said VL region has at least 90%, atleast 95%, at least 97%, or at least 99% amino acid sequence identity tothe amino acid sequence as set forth in the VL sequences selected fromthe group consisting of; a) a VL sequence as set forth in SEQ ID NO:10;b) a VL sequence as set forth in SEQ ID NO:11; and c) a VL sequence asset forth in SEQ ID NO:12.
 4. The antibody according to any one of thepreceding claims, wherein said VH region is selected from the groupconsisting of; a) a VH sequence as set forth in SEQ ID NO:6; b) a VHsequence as set forth in SEQ ID NO:8; c) a VH sequence as set forth inSEQ ID NO:7; and d) a VH sequence as set forth in SEQ ID NO:9.
 5. Theantibody according to any one of the preceding claims, wherein said VLregion is selected from the group consisting of; a) a VL sequence as setforth in SEQ ID NO:10; b) a VL sequence as set forth in SEQ ID NO:11;and c) a VL sequence as set forth in SEQ ID NO:12.
 6. The antibodyaccording to any one of the preceding claims, wherein said VH and VLregions are selected from the group consisting of; a) a VH sequence asset forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ IDNO:10; b) a VH sequence as set forth in SEQ ID NO:8, and a VL sequenceas set forth in SEQ ID NO:10; c) a VH sequence as set forth in SEQ IDNO:9, and a VL sequence as set forth in SEQ ID NO:10; d) a VH sequenceas set forth in SEQ ID NO:6, and a VL sequence as set forth in SEQ IDNO:11; e) a VH sequence as set forth in SEQ ID NO:6, and a VL sequenceas set forth in SEQ ID NO:12; f) a VH sequence as set forth in SEQ IDNO:7, and a VL sequence as set forth in SEQ ID NO:10; g) a VH sequenceas set forth in SEQ ID NO:7, and a VL sequence as set forth in SEQ IDNO:11; h) a VH sequence as set forth in SEQ ID NO:7, and a VL sequenceas set forth in SEQ ID NO:12; i) a VH sequence as set forth in SEQ IDNO:8, and a VL sequence as set forth in SEQ ID NO:11; j) a VH sequenceas set forth in SEQ ID NO:8, and a VL sequence as set forth in SEQ IDNO:12; k) a VH sequence as set forth in SEQ ID NO:9, and a VL sequenceas set forth in SEQ ID NO:11; and l) a VH sequence as set forth in SEQID NO:9, and a VL sequence as set forth in SEQ ID NO:12.
 7. The antibodyaccording to any one of the preceding claims, wherein said bindingregion comprises a VH sequence and a VL sequence selected from the groupconsisting of; a) a VH sequence as set forth in SEQ ID NO:6, and a VLsequence as set forth in SEQ ID NO:10; b) a VH sequence as set forth inSEQ ID NO:8, and a VL sequence as set forth in SEQ ID NO:10; and c) a VHsequence as set forth in SEQ ID NO:9, and a VL sequence as set forth inSEQ ID NO:10.
 8. The antibody according to any one of the precedingclaims, wherein the antibody is a humanized antibody.
 9. The antibodyaccording to claim 1, wherein the antibody is a chimeric antibody. 10.The antibody according to any one of the preceding claims, wherein theantibody is a full-length antibody.
 11. The antibody according to anyone of the preceding claims, wherein said antibody comprises an Fcregion comprising a first and a second immunoglobulin heavy chain. 12.The antibody according to any one of the preceding claims, wherein saidfirst and said second heavy chains are of an isotype selected from thegroup consisting of IgG1, IgG2, IgG3, and IgG4.
 13. The antibodyaccording to any one of the preceding claims, wherein said antibodycomprises an Fc region which has been modified so that binding of C1q tosaid antibody is reduced compared to a wild-type antibody by at least70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%,wherein C1q binding is determined by ELISA.
 14. The antibody accordingto any one of the preceding claims, wherein said antibody comprises anFc region which has been modified so that said antibody mediates reducedFc-mediated T-cell proliferation compared to a wild-type antibody by atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 99% or 100%, wherein said T-cell proliferation is measured in aperipheral blood mononuclear cell (PBMC)-based functional assay.
 15. Theantibody according to any one of the preceding claims, wherein saidantibody comprises an Fc region which has been modified so that saidantibody reduces Fc-mediated CD69 expression by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% whencompared to a wild-type antibody wherein said Fc-mediated CD69expression is determined in a PBMC-based functional assay.
 16. Theantibody according to any one of the preceding claims, wherein saidantibody comprises a first and a second immunoglobulin heavy chain,wherein in at least one of said first and second immunoglobulin heavychains one or more amino acids in the positions corresponding topositions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain,are not L, L, D, N, and P, respectively.
 17. The antibody according toclaim 16, wherein in at least one of said first and second heavy chainsthe amino acid in the position corresponding to position D265 in a humanIgG1 heavy chain, is not D.
 18. The antibody according to claim 16,wherein in at least one of said first and second heavy chains the aminoacid in the position corresponding to position N297 in a human IgG1heavy chain, is not N.
 19. The antibody according to claim 16, whereinin at least one of said first and second heavy chains the amino acids inthe positions corresponding to positions L234 and L235 in a human IgG1heavy chain, are not L and L, respectively.
 20. The antibody accordingto any of claims 16 and 19, wherein in at least one of said first andsecond heavy chains the amino acids in the positions corresponding topositions L234 and L235 in a human IgG1 heavy chain, are F and E; or Aand A, respectively.
 21. The antibody according to claim 20, wherein inat least one of said first and second heavy chains the amino acids inthe positions corresponding to positions L234 and L235 in a human IgG1heavy chain, are F and E, respectively.
 22. The antibody according toclaim 20, wherein in at least one of said first and second heavy chainsat least the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain, are A and A, respectively.23. The antibody according to any of claims 1 to 16, wherein in at leastone of said first and second heavy chains the amino acids in thepositions corresponding to positions L234, L235, and D265 in a humanIgG1 heavy chain, are not L, L, and D, respectively.
 24. The antibodyaccording to claim 23, wherein in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A,A, and A, respectively.
 25. The antibody according to claim 24, whereinin at least one of said first and second heavy chains the amino acids inthe positions corresponding to positions L234, L235, and D265 in a humanIgG1 heavy chain, are F, E, and A, respectively.
 26. The antibodyaccording to claim 24, wherein in at least one of said first and secondheavy chains the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A,respectively.
 27. The antibody according to claim 16, wherein in atleast one of said first and second heavy chains the amino acids in thepositions corresponding to positions L234, L235, D265, N297, and P331 ina human IgG1 heavy chain, are F, E, A, Q, and S, respectively.
 28. Theantibody according to any one of the preceding claims, wherein saidantibody comprises a light chain (LC), and wherein the amino acid in theposition corresponding to position T41 in the lambda light chain of SEQID NO:10 is not T.
 29. The antibody according to claim 28, wherein theamino acid in the position corresponding to position T41 in the lambdalight chain of SEQ ID NO:10 is K.
 30. The antibody according to any oneof the preceding claims 1 to 28, wherein the amino acid in the positioncorresponding to position F10 in the lambda light chain of SEQ ID NO:10is not F, and wherein one or more of the amino acid positionscorresponding to the positions T41, K55, and L97 in the lambda lightchain of SEQ ID NO:10 are not T, K and L, respectively.
 31. The antibodyaccording to claim 30, wherein the amino acids in the positionscorresponding to positions F10, T41, K55, and L97 in the lambda lightchain of SEQ ID NO:10 are not F, T, K and L, respectively.
 32. Theantibody according to claim 31, wherein the amino acids in the positionscorresponding to positions F10, T41, K55, and L97 in the lambda lightchain of SEQ ID NO:10 are L, K, N, and H, respectively.
 33. The antibodyaccording to any one of the preceding claims 1 to 28, wherein the aminoacids in the positions corresponding to positions R23 and A35 are not Rand A, respectively.
 34. The antibody according to claim 33, wherein theamino acids in the positions corresponding to positions R23 and A35 areA and P, respectively.
 35. The antibody according to claim 33, whereinthe amino acids in the positions corresponding to positions F10, R23,A35, R47, D71, A82, D83, S86, 187, and F89 in the lambda light chain ofSEQ ID NO:10 are not F, R, A, R, D, A, D, S, I, and F, respectively. 36.The antibody according to claim 35, wherein the amino acids in thepositions corresponding to positions F10, R23, A35, R47, D71, A82, D83,S86, 187, and F89 in the lambda light chain of SEQ ID NO:10 are L, A, P,T, G, P, E, A, E, and Y, respectively.
 37. The antibody according to anyone of the preceding claims 1 to 36, wherein (i) the amino acid in theposition corresponding to position F10 in the lambda light chain of SEQID NO:10 is not F, or (ii) the amino acid in the position correspondingto position K55 in the lambda light chain of SEQ ID NO:10 is not K, or(iii) the amino acid in the position corresponding to position F10 inthe lambda light chain of SEQ ID NO:10 is not F, and the amino acid inthe position corresponding to position K55 in the lambda light chain ofSEQ ID NO:10 is not K.
 38. The antibody according to claim 37, wherein(i) the amino acid in the position corresponding to position F10 in thelambda light chain of SEQ ID NO:10 is L, or (ii) the amino acid in theposition corresponding to position K55 in the lambda light chain of SEQID NO:10 is N, or (iii) the amino acid in the position corresponding toposition F10 in the lambda light chain of SEQ ID NO:10 is L, and theamino acid in the position corresponding to position K55 in the lambdalight chain of SEQ ID NO:10 is N.
 39. The antibody according to any oneof the preceding claims 1 to 38, wherein said antibody comprises aconstant heavy chain (HC) and a constant light chain (LC), wherein thepositions corresponding to positions L234, L235, and D265 in the humanIgG1 heavy chain of SEQ ID NO:15 are F, E, and A, respectively, andwherein the position corresponding to F405 in in the human IgG1 heavychain of SEQ ID NO:15 is L, and wherein (i) the positions correspondingto positions F10, T41, K55, and L97 in the lambda light chain of SEQ IDNO:10 are L, K, N, and H, respectively, or (ii) the positioncorresponding to position T41 in the lambda light chain of SEQ ID NO:10is K.
 40. A bispecific antibody comprising a first binding region of anantibody according to any one of claims 1 to 39, and a second bindingregion which binds a different target than said first antigen bindingregion.
 41. The bispecific antibody according to claim 40, wherein saidantibody comprises a first and a second heavy chain.
 42. The bispecificantibody according to claim 41, wherein a) said bispecific antibodycomprises an Fc region modified according to any one of claims 13 to 15;or b) at least one of said first and second heavy chains comprise one ormore amino acids modified as defined in any of claims 16 to
 39. 43. Thebispecific antibody according to any one of claims 40 to 42, whereineach of said first and second heavy chain comprises at least a hingeregion, a CH2 and CH3 region, wherein in said first heavy chain at leastone of the amino acids in the positions corresponding to a positionsselected from the group consisting of T366, L368, K370, D399, F405,Y407, and K409 in a human IgG1 heavy chain has been substituted, and insaid second heavy chain at least one of the amino acids in the positionscorresponding to a position selected from the group consisting of T366,L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain hasbeen substituted, and wherein said first and said second heavy chainsare not substituted in the same positions.
 44. The bispecific antibodyaccording to claim 43, wherein the amino acid in the positioncorresponding to F405 in a human IgG1 heavy chain is L in said firstheavy chain, and the amino acid in the position corresponding to K409 ina human IgG1 heavy chain is R in said second heavy chain, or vice versa.45. The bispecific antibody according to any one of claims 40 to 44,wherein said first binding region is according to any of claims 1 to 7,and said second binding region binds a different target than said firstbinding region.
 46. The bispecific antibody according to claim 45,wherein the second binding region binds to human HER2 or human CD20. 47.The bispecific antibody according to claim 46, wherein the secondbinding region binds to human CD20.
 48. The bispecific antibodyaccording to claim 47, wherein the second binding region binding tohuman CD20 comprises: (i) the VH CDR1 region of SEQ ID NO:34, the VHCDR2 region of SEQ ID NO:35, the VH CDR3 region of SEQ ID NO:36, the VLCDR1 region of SEQ ID NO:37, the VL CDR2 region of DAS, and the VL CDR3region of SEQ ID NO:38, (ii) the VH CDR1 region of SEQ ID NO: 41, the VHCDR2 region of SEQ ID NO:42, the VH CDR3 region of SEQ ID NO:43, the VLCDR1 region of SEQ ID NO:44, the VL CDR2 region of DAS, and the VL CDR3region of SEQ ID NO:45, (iii) the VH CDR1 region of SEQ ID NO:48, the VHCDR2 region of SEQ ID NO:49, the VH CDR3 region of SEQ ID NO:50, the VLCDR1 region of SEQ ID NO:51, the VL CDR2 region of DAS, and the VL CDR3region of SEQ ID NO:52, or (iv) the VH CDR1 region of SEQ ID NO:55, theVH CDR2 region of SEQ ID NO:56, the VH CDR3 region of SEQ ID NO:57, theVL CDR1 region of SEQ ID NO:58, the VL CDR2 region of DAS, and the VLCDR3 region of SEQ ID NO:59.
 49. The bispecific antibody according toclaim 47 or 48, wherein the second binding region binding to human CD20comprises: (i) a VH sequence which has at least 90%, at least 95%, atleast 97%, or at least 99% amino acid sequence identity to the aminoacid sequence as set forth in SEQ ID NO:29, and a VL sequence which hasat least 90%, at least 95%, at least 97%, or at least 99% amino acidsequence identity to the amino acid sequence as set forth in SEQ IDNO:30, (ii) a VH sequence which has at least 90%, at least 95%, at least97%, or at least 99% amino acid sequence identity to the amino acidsequence as set forth in SEQ ID NO:39, and a VL sequence which has atleast 90%, at least 95%, at least 97%, or at least 99% amino acidsequence identity to the amino acid sequence as set forth in SEQ IDNO:40, (iii) a VH sequence which has at least 90%, at least 95%, atleast 97%, or at least 99% amino acid sequence identity to the aminoacid sequence as set forth in SEQ ID NO:46, and a VL sequence which hasat least 90%, at least 95%, at least 97%, or at least 99% amino acidsequence identity to the amino acid sequence as set forth in SEQ IDNO:47, or (iv) a VH sequence which has at least 90%, at least 95%, atleast 97%, or at least 99% amino acid sequence identity to the aminoacid sequence as set forth in SEQ ID NO:53, and a VL sequence which hasat least 90%, at least 95%, at least 97%, or at least 99% amino acidsequence identity to the amino acid sequence as set forth in SEQ IDNO:54.
 50. The bispecific antibody according to claim 49, wherein thesecond binding region binding to human CD20 comprises: (i) the VHsequence of SEQ ID NO: 29 and the VL sequence of SEQ ID NO:30, (ii) theVH sequence of SEQ ID NO:39, and the VL sequence of SEQ ID NO:40, (iii)the VH sequence of SEQ ID NO:46, and the VL sequence of SEQ ID NO:47, or(iv) the VH sequence of SEQ ID NO:53, and the VL sequence of SEQ IDNO:54.
 51. A nucleic acid construct encoding one or more amino acidsequences set out in Table
 1. 52. An expression vector comprising (i) anucleic acid sequence encoding a heavy chain sequence of a humanized orchimeric antibody according to any one of claims 1 to 50; (ii) a nucleicacid sequence encoding a light chain sequence of a humanized or chimericantibody according to any one of claims 1 to 50; or (iii) both (i) and(ii).
 53. A host cell comprising an expression vector of claim
 52. 54.The host cell according to claim 53, wherein said host cell is arecombinant eukaryotic, recombinant prokaryotic, or recombinantmicrobial host cell.
 55. A composition comprising an antibody accordingto any one of claims 1 to 39 or a bispecific antibody according to anyone of claims 40 to
 50. 56. A pharmaceutical composition comprising theantibody according to any one of claims 1 to 39 or bispecific antibodyaccording to any one of claims 40 to 50 and a pharmaceutical acceptablecarrier.
 57. The antibody according to any one of claims 1 to 39, saidbispecific antibody according to any one of claims 40 to 50, saidcomposition according to claim 55, or said pharmaceutical compositionaccording to claim 56 for use as a medicament.
 58. The antibodyaccording to any one of claims 1 to 39, said bispecific antibodyaccording to any one of claims 40 to 50, said composition according toclaim 55, or said pharmaceutical composition according to claim 56, foruse in the treatment of a disease.
 59. A method of treatment of adisease comprising administering said antibody according to any one ofclaims 1 to 39, said bispecific antibody according to any one of claims40 to 50, said composition according to claim 55, or said pharmaceuticalcomposition according to claim 56, to a subject in need thereof.
 60. Theuse or method according to any one of claims 57 to 59, wherein thedisease is cancer, an infectious disease, or an autoimmune disease. 61.A method of diagnosing a disease characterized by involvement oraccumulation of CD3-expressing cells, comprising administering saidantibody according to any one of claims 1 to 39, said bispecificantibody according to any one of claims 40 to 50, said compositionaccording to claim 55, or said pharmaceutical composition according toclaim 56 to a subject, optionally wherein said antibody or saidbispecific antibody is labeled with a detectable agent.
 62. A method forproducing an antibody according to any one of claims 1 to 39, or abispecific antibody according to any one of claims 40 to 50, comprisingthe steps of a) culturing a host cell according to any one of claims 53to 54; and b) purifying said antibody from the culture media.
 63. Adiagnostic composition comprising an antibody according to any one ofclaims 1 to 39, or a bispecific antibody according to any one of claims40 to
 50. 64. A method for detecting the presence of CD3 antigen, or acell expressing CD3, in a sample comprising the steps of; a) contactingthe sample with an antibody according to any one of claims 1 to 39 or abispecific antibody according to any one of claims 40 to 50, underconditions that allow for formation of a complex between said antibodyor bispecific antibody and CD3; and b) analyzing whether a complex hasbeen formed.
 65. A kit for detecting the presence of CD3 antigen, or acell expressing CD3, in a sample comprising i) an antibody according toany one of claims 1 to 39, or a bispecific antibody according to any oneof claims 40 to 50; and ii) instructions for use of said kit.
 66. Ananti-idiotypic antibody which binds to an antibody of any one of claims1 to 39.